Molecular biologic comparison of new bone formation and resorption on microrough and smooth bioactive glass microspheres

In a recent in vitro study, chemical microroughening of a bioactive glass surface was shown to enhance attachment of MG-63 osteoblastic cells to glass. The current study was designed to delineate the effects of microroughening on the gene expression patterns of bone markers during osteogenesis and new bone remodeling on bioactive glass surface in vivo. With the use of a rat model of paired comparison, a portion of the medullary canal in the proximal tibia was evacuated through cortical windows and filled with microroughened or smooth bioactive glass microspheres. The primary bone-healing response and subsequent remodeling were analyzed at 1, 2, and 8 weeks, respectively, by radiography, pQCT, histomorphometry, BEI-SEM, and molecular biologic analyses. The expression of various genes for bone matrix components (type I collagen, osteocalcin, osteopontin, osteonectin) and proteolytic enzymes (cathepsin K, MMP-9) were determined by Northern analysis of the respective mRNAs. Paired comparison showed significant differences in the mRNAs levels for specific bone matrix components at 2 weeks: osteopontin was significantly higher (p =.01) and osteonectin significantly lower (p =.05) in bones filled with microroughened microspheres than in those filled with smooth microspheres. Bones filled with microrough microspheres also showed significantly increased ratios of cathepsin K and MMP-9 (both markers of osteoclastic resorption) to type I collagen (p =.02 and p =.02, respectively) at 2 weeks and a significantly increased expression of MMP-9 at 8 weeks (p =.05). The pQCT, histomorphometric, and BEI-SEM analyses revealed no significant differences in the pattern of bone-healing response. Based on these results, microroughening of a bioactive glass surface could trigger temporal changes in the expression of specific genes especially by promoting the resorption part of new bone-remodeling processes. Future studies are needed to evaluate if the observed changes of gene expression are directly related to the microrough surface of any biomaterial or are biomaterial specific.     

Characterization of microrough bioactive glass surface: surface reactions and osteoblast responses in vitro

The current study characterized the in vitro surface reactions of microroughened bioactive glasses and compared osteoblast cell responses between smooth and microrough surfaces. Three different bioactive glass compositions were used and surface microroughening was obtained using a novel chemical etching method. Porous bioactive glass specimens made of sintered microspheres were immersed in simulated body fluid (SBF) or Tris solutions for 1, 6, 24, 48, or 72 h, and the formation of reaction layers was studied by means of a scanning electron microscope/energy dispersive X-ray analysis (SEM/EDXA). Cell culture studies were performed on bioactive glass disks to examine the influence of surface microroughness on the attachment and proliferation of human osteoblast-like cells (MG-63). Cell attachment was evaluated by means of microscopic counting of in situ stained cells. Cell proliferation was analyzed with a nonradioactive cell proliferation assay combined with in situ staining and laser confocal microscopy. The microroughening of the bioactive glass surface increased the rate of the silica gel layer formation during the first hours of the immersion. The formation of calcium phosphate layer was equal between control and microroughened glass surfaces. In cell cultures on bioactive glass, the microrough surface enhanced the attachment of osteoblast-like cells but did not have an effect on the proliferation rate or morphology of the cells as compared with smooth glass surface. In conclusion, microroughening significantly accelerated the early formation of surface reactions on three bioactive glasses and had a positive effect on initial cell attachment.     

Osteoblast responses to tape-cast and sintered bioactive glass ceramics

The advantage of tape-cast bioactive glasses lies in the manufacturing procedure, which allows the build-up of layers and, therefore, the production of complex shapes. This, therefore, has applications to tissue engineering, where specific shapes are required such as repair of craniofacial defects. The bioactivity of tape-cast discs sintered at temperatures ranging from 800 degrees C to 1000 degrees C and for 3 or 6 h was analyzed by FTIR. Tape-cast discs were used to culture primary human osteoblasts, and cell attachment, cell death, collagen production, nodule formation, and mineralization were studied. These responses were dependent upon Si and Na release profiles of the tape-cast discs, and development of the hydroxyapatite layer.     

Acceleration of apatite nucleation on microrough bioactive titanium for bone-replacing implants

The viability of a new two-step method for obtaining bioactive microrough titanium surfaces for bone replacing implants has been evaluated. The method consists of (1) Grit blasting on titanium surface to roughen it; and (2) Thermo-chemical treating to obtain a bioactive surface with bone-bonding ability by means of nucleating and growing an apatite layer on the treated surface of the metal. The aim of this work is to evaluate the effect of surface roughness and chemical composition of the grit-blasting particles on the ability of the surfaces of nucleating and growing a homogeneous apatite layer. The determination and kinetics of the nucleation and growing of the apatite layer on the surfaces has mainly been studied with environmental scanning electron microscopy (ESEM) and grazing-incidence X-ray diffractometry. The results show that Al(2)O(3)-blasted and thermochemically-treated titanium surfaces accelerates nucleation of the apatite, whereas SiC-blasted and thermochemically-treated titanium surfaces inhibits apatite nucleation, compared with the well studied polished and thermochemically-treated titanium surfaces. The acceleration of the apatite nucleation on the Al(2)O(3)-blasted microrough titanium surfaces is because concave parts of the microroughness that are obtained during grit blasting provides to the rough and bioactive surfaces with a chemical- and electrostatic-favored situation for apatite nucleation. This consists of a high density of surface negative charges (also assisted by the nanoroughness of the surface obtained after the thermochemical treatment) and an increased concentration of the Ca(2+)-ions of the fluid, which have a limited mobility at the bottom of the concave parts.     

On the microstructure of biocomposites sintered from Ti, HA and bioactive glass

Sintering reactions and fine structures of the biocomposites prepared from powder mixtures of titanium ( alpha -Ti), hydroxyapatite (HA) and bioactive glass (BG) (SiO2-CaO-P2O5-B2O3-MgO-TiO2-CaF2) were investigated by X-ray diffraction and transmission electron microscopy. The results showed that complex reactions among the starting materials mainly depended on the initial Ti/HA ratios as well as the sintering temperatures. And the reaction could be expressed by the following illustrative equation: Ti+Ca10(PO4)6(OH)2-->CaTiO3+CaO+TixPy+(Ti2O)+(Ca4P2O9)+H2O.     

Effects of bioactive glass and beta-TCP containing three-dimensional laser sintered polyetheretherketone composites on osteoblasts in vitro

Because of their excellent physical properties nonresorbable thermoplastic polymers have become more important for the field of reconstructive surgery. Aim of the present study was to investigate the effects of laser sintered polyetheretherketone (PEEK) with incorporated osteoconductive and bioactive bone substitution materials on osteoblasts in vitro. Human osteoblasts (hFOB 1.19) were seeded onto laser sintered PEEK samples containing nano-sized carbon black, beta-tricalciumphosphate (beta-TCP), and bioactive glass 45S5. Osteoblasts were investigated for cell viability, cell proliferation and cell morphology. A constant proliferation of osteoblasts could be observed on all samples with the highest values for bioactive glass containing samples at day 7 (OD 1.76 +/- 0.22) and day 14 (OD 3.75 +/- 0.31) and lowest values for beta-TCP containing probes throughout the study compared with the PEEK pure control group. Highest cell viability was observed for Bioglass containing probes (95.5 +/- 3.32)% whereas osteoblasts seeded on beta-TCP containing probes showed reduced viability (84.4 +/- 4.32)%. Laser sintered PEEK implants seem to be attractive candidates for use as bone substitutes for reconstructive surgery because of their biocompatibility, individual shape, and the possibility of compounding bioinert polymer powder with osteoconductive and bioactive materials which might benefit bone formation in vivo.     

Combined effect of BMP-2 gene transfer and bioactive glass microspheres on enhancement of new bone formation

Adenovirus-mediated recombinant human BMP-2 (RAdBMP-2) gene transfer has been found to have significant osteoinductive properties. The hypothesis of the current study was that bioactive glass surface could provide favorable osteoconductive conditions for cellular action of osteoinductive RAdBMP-2 gene transfer. In the rat proximal tibia, a portion of the medullary cavity was evacuated and filled with bioactive glass microspheres and injected with adenovirus carrying the human BMP-2 gene (BG/RAdBMP-2). Control defects filled with BG microspheres were injected with adenovirus carrying the LacZ reporter gene (BG/RAdLacZ) or saline (BG). Empty control defects were also used. Bone healing response was analyzed at 4 days, and at 2 and 8 weeks by radiography, peripheral quantitative computed tomography (pQCT), histomorphometry, and backscattered electron imaging of scanning electron microscopy (BEI-SEM) equipped with energy dispersive X-ray analysis (EDXA). In empty controls, the amount of intramedullary new bone peaked at 2 weeks, whereas defects filled with bioactive glass with and without RAdBMP-2 gene transfer showed a constant time-related increase of intramedullary new bone. At 8 weeks, there was significantly more new bone in defects treated with BG and RAdBMP-2 than in defects left to heal without filling (p < 0.001). Compared with the other controls (BG only or BG/RAdLacZ), the difference was not significant. In the current model, the osteopromotive effect of bioactive glass microspheres appears synergistic with the osteoinductive action of BMP-2 gene transfer, or one overshadows the other, as no additive effect was observed.     

Calcium phosphate formation at the surface of bioactive glass in vitro

The calcium phosphate formation at the surface of bioactive glass was studied in vitro. Glass rods and grains were immersed in different aqueous solutions and studied by means of scanning electron microscopy and energy dispersive x-ray analysis. Surface morphological changes and weight loss of corroded grains were monitored. In-depth compositional profiles were determined for rods immersed in the different solutions. The solutions used were tris-buffer (tris-hydroxymethylaminomethane + HCl), tris-buffer prepared using citric acid (tris-hydroxymethylaminomethane + C6H8O7.H2O), and a simulated body fluid, SBF, containing inorganic ions close in concentration to those in human blood plasma. It was found that the calcium phosphate formation at the surface of bioactive glass in vitro proceeds in two stages. When immersing the glass in tris or in SBF a Ca,P-rich surface layer forms. This accumulation takes place within the silica structure. Later, apatite crystals forming spherulites appear on the surface. The Ca/P-ratio of initially formed calcium phosphate was found to be about unity. It is proposed that this is due to bonding of phosphate to a silica gel. The surface is stabilized, i.e., leaching is retarded, by the rapid Ca,P-accumulation within the silica structure before apatite crystals are observed on the surface. It is proposed that the initially formed calcium phosphate is initiated within the silica gel. The crystallizing surface provides nucleation sites for extensive apatite formation on the glass surface. In the presence of citrate no Ca,P-accumulation occur at the glass surface, but soluble Ca-citrate complexes form. By comparing the weight loss during corrosion in tris with that in the calcium and phosphate containing SBF, it is possible to establish whether the glass can induce apatite formation at its surface or not.     

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.     

Biologic significance of surface microroughing in bone incorporation of porous bioactive glass implants

A novel chemical etching method was recently developed to create a controlled microrough surface on porous bioactive glass implants. Our earlier in vitro studies showed enhanced attachment of osteoblast-like MG63 cells on a microrough bioactive glass surface. The purpose of our current study was to confirm the in vivo significance of surface microroughening for bone bonding of bioactive glass. Porous bioactive glass cones made of sintered microspheres were surgically implanted in the anterior cortex of rabbit femurs. Peripheral quantitative computed tomography (pQCT), biomechanical push-out testing, histomorphometry, and electron microscopy (BEI-SEM) were used to analyze bone ingrowth and osseointegration at 7, 10, 14, 28, 56, and 84 days after implantation. The results showed that microroughening of the bioactive glass surface significantly enhanced the bone-bonding response of the biomaterial. The positive response was seen in one of the three bioactive glass compositions studied. The affinity index of new bone on the glass surface was significantly (p = 0.02) increased with a trend (p = 0.10) toward improved mechanical incorporation. New bone formation was dependent on the glass composition, and it was found to occur not only through the mechanism of bone ingrowth but also based on in situ osteogenesis within implant interstices. Based on these results, the procedure of microroughening could enhance the osteopromotive properties of certain bioactive glass compositions.     

Intact surface of bioactive glass S53P4 is resistant to osteoclastic activity

Bioactive glass reacts with body fluids and is gradually dissolved in tissues and in cell cultures. We investigated whether osteoclasts contribute to this process, by culturing newborn rat bone-marrow cells containing osteoclasts on polished bioactive glass plates (glass S53P4). The cultures were inspected at days 1-5 and stained for alkaline phosphatase (ALP) to demonstrate osteoblasts and for tartrate resistant acid phosphatase (TRAP) to visualize osteoclasts. Nonosteoclastic cells proliferated several-fold both on bioactive glass and on plastic, whereas osteoclasts and their precursors matured into multicellular giant cells and degenerated. Most cells on bioactive glass became ALP-positive, whereas on plastic the majority of cells remained ALP-negative. Osteoclasts survived on bioactive glass for 4-5 days, whereas on plastic they degenerated and disappeared after 3 days. Condensed nuclei indicating apoptosis were detected both in degenerating osteoclasts and osteoblasts. The surface of the bioactive glass reacted rapidly forming rounded pits, erosions, and cracks within 24 h in areas occupied by osteoblasts. Light microscopy and scanning electron micrographs demonstrated, however, a smooth surface below the cytoplasm of osteoclasts. This indicates that when applied on an intact bioactive glass surface, osteoclasts were unable to dissolve the glass material within this culture period.     

Laser cladding of bioactive glass coatings

Laser cladding by powder injection has been used to produce bioactive glass coatings on titanium alloy (Ti6Al4V) substrates. Bioactive glass compositions alternative to 45S5 Bioglass^® were demonstrated to exhibit a gradual wetting angle–temperature evolution and therefore a more homogeneous deposition of the coating over the substrate was achieved. Among the different compositions studied, the S520 bioactive glass showed smoother wetting angle–temperature behavior and was successfully used as precursor material to produce bioactive coatings. Coatings processed using a Nd:YAG laser presented calcium silicate crystallization at the surface, with a uniform composition along the coating cross-section, and no significant dilution of the titanium alloy was observed. These coatings maintain similar bioactivity to that of the precursor material as demonstrated by immersion in simulated body fluid.     

Surface characterization of silver-doped bioactive glass

A bioactive glass belonging to the system SiO(2)-CaO-Na(2)O was doped with silver ions by ion exchange in molten salts as well as in aqueous solution. The ion exchange in the solution was done to check if it is possible to prepare an antimicrobial material using a low silver content. The doped glass was characterized by means of X-ray diffraction, SEM observation, EDS analysis, bioactivity test (soaking in a simulated body fluid), leaching test (GFAAS analyses) and cytotoxicity test. It is demonstrated that these surface silver-doped glasses maintain, or even improve, the bioactivity of the starting glass. The measured quantity of released silver into simulated body fluid compares those reported in literature for the antibacterial activity and the non-cytotoxic effect of silver. Cytotoxicity tests were carried out to understand the effect of the doped surfaces on osteogenic cell adhesion and proliferation.     

Biomimetic and nanostructured hybrid bioactive glass

Inspired by nature's toughening mechanisms, we designed a new polyhedral oligomeric silsesquioxane (POSS)-derived hybrid glass (PHG) that has covalent interactions on the molecular scale between the inorganic POSS cage and organic phase. These features allow “elastic deformation” of the inorganic POSS cage in limited scale. The final product is a bulk hybrid material with toughness (3.56 ± 0.25 MPa·m^1/2) similar to natural bone (2.4–5.3 MPa·m^1/2). PHG exhibited excellent bioactivity by promoting the formation of plate-like hydroxyapatite on its surface in simulated body fluid and showed good cell adhesion. PHG also can be a platform to guide adipose tissue-derived mesenchymal stem cells differentiation and mineralization. The key structural features of this material can be used to guide the design of bio-inspired composites with unique toughness, which would be of great benefit to hard tissue engineering.     

Adhesion of new bioactive glass coating.

A valuable alternative to the existing biomedical implant coatings is a bioactive glass (BAG) coating that is produced by reactive plasma spraying. A mechanical performance requirement that is of the utmost importance is the adhesion strength of the coating. Considering the application as dental implant, a new adhesion test (shear test), which was close to the service conditions, was designed. A Ti6Al4V rod (3 mm) with a sprayed BAG coating of 50 microm was glued with an epoxy glue to a hollow cylindrical counterpart and was used as such in the tensile machine. This test was evaluated by finite element analysis (FEA). Preliminary experiments showed that a conversion from shear to tensile adhesion strength is possible by using the Von Mises criterion (sigma = 3(1/2)tau), indicating that thin coatings of brittle materials can behave as a ductile material. The new coating technique was proved to produce a high quality coating with an adhesion strength of 40.1 +/- 4.8 MPa in shear and 69.4 +/- 8.4 MPa in tension. The FEA revealed that no one homogeneously distributed shear stress is present but several nonhomogeneously distributed stress components (shear and tensile) are present in the coating. This analysis indicated that real service conditions are much more complicated than standard adhesion tests.     

Bioactivity of electro-thermally poled bioactive silicate glass

A 45S5 bioactive glass (nominal composition: 46.1 mol.% SiO₂, 2.6 mol.% P₂O₅, 26.9 mol.% CaO, 24.4 mol.% Na₂O) was electrothermally poled by applying voltages up to 750 V for 45 min at 200 °C, and the thermally stimulated depolarization currents (TSDCs) were recorded. Changes in chemical composition and electrical properties after poling were investigated by TSDC measurements, impedance spectroscopy and scanning electron microscopy with energy dispersive X-ray spectroscopy (SEM/EDX). The poling led to the formation of interfacial layers underneath the surface in contact with the electrodes. Under the positive electrode, the layer was characterized by Na⁺ ion depletion and by a negative charge density, and the layer was more resistive than the bulk. The influence of poling on the bioactivity was studied by immersion of samples in simulated body fluid (SBF) with subsequent cross-sectional SEM/EDX and X-ray diffraction analysis. It was found that poling leads to morphological changes in the silica-rich layer and to changes in the growth rate of amorphous calcium phosphate and bone-like apatite on the glass surface. The bone-like apatite layer under the positive electrode was slightly thicker than that under the negative electrode.     

Dose-dependent behavior of bioactive glass dissolution.

The effect of glass dosage (0.001 g ml(-1) to 0.015 g ml(-1)) on the in vitro dynamic dissolution behavior of melt-derived 45S5 and sol-gel-derived 58S bioactive glasses, in simulated body fluid (SBF) at 37 degrees C, was evaluated. These glasses differ significantly in texture, especially the specific surface area and porosity, as a result of differences in manufacturing route. The concentrations of elements (Si, Ca, P, and Na) leached from the glasses into the dissolution medium, from 1 to 22 h, were evaluated with the use of induced coupled plasma analysis (ICP). The reacted powders were analyzed with the use of FTIR to observe the formation of a hydroxycarbonate apatite layer on the surface. The results show that the rate of HCA formation on both gel- and melt-derived bioactive glass powders in vitro depends on the concentration of the powders in solution. This result must be taken into account when carrying out in vitro cell-culture studies to simulate conditions in vivo and in experiments using extracts of the bioactive glass powders.     

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.     

Preparation and bioactive properties of novel bone-repair bionanocomposites based on hydroxyapatite and bioactive glass nanoparticles

Bionanocomposites based on ceramic nanoparticles and a biodegradable porous matrix represent a promising strategy for bone repair applications. The preparation and bioactive properties of bionanocomposites based on hydroxyapatite (nHA) and bioactive glass (nBG) nanoparticles were presented. nHA and nBG were synthesized with nanometric particle size using sol-gel/precipitation methods. Composite scaffolds were prepared by incorporating nHA and nBG into a porous alginate (ALG) matrix at different particle loads. The ability of the bionanocomposites to induce the crystallization of the apatite phase from simulated body fluid (SBF) was systematically evaluated using X-ray diffraction (XRD), scanning electron microscopy with energy dispersive X-ray analysis, and Fourier transform infrared spectroscopy. Both nHA/ALG and nBG/ALG composites were shown to notably accelerate the process of crystallization and growth of the apatite phase on the scaffold surfaces. For short immersion times in SBF, nBG (25%)-based nanocomposites induced a higher degree of apatite crystallization than nHA (25%)-based nanocomposites, probably due to the more reactive nature of the BG particles. Through a reinforcement effect, the nanoparticles also improve the mechanical properties and stability in SBF of the polymer scaffold matrix. In addition, in vitro biocompatibility tests demonstrated that osteoblast cells are viable and adhere well on the surface of the bionanocomposites. These results indicate that nHA- and nBG-based bionanocomposites present potential properties for bone repair applications, particularly oriented to accelerate the bone mineralization process.     

Development of bioactive and biodegradable chitosan-based injectable systems containing bioactive glass nanoparticles

There is increasing interest in the development of new tissue engineering strategies to deliver cells and bioactive agents encapsulated in a biodegradable matrix through minimally invasive procedures. The present work proposes to combine chitosan-beta-glycerophosphate salt formulations with bioactive glass nanoparticles in order to conceive novel injectable thermo-responsive hydrogels for orthopaedic reconstructive and regenerative medicine applications. The initial rheological properties and the gelation points of the developed organic-inorganic in situ thermosetting systems were revealed to be adequate for intracorporal injection. In vitro bioactivity tests, using incubation protocols in simulated body fluid (SBF), allowed the observation of bone-like apatite formation in the hydrogel formulations containing bioactive nanoparticles. The density of the apatite formed increased with increasing bioactive glass content and soaking time in SBF. These results indicate that the stimuli-responsive hydrogels could potentially be used as temporary injectable scaffolds in bone tissue engineering applications.