45S5 Bioactive Glass Research Articles

Variation of the properties of sol–gel synthesized bioactive glass 45S5 in organic and inorganic acid catalysts

Sol–gel synthesis of BG 45S5 using organic acetic acid as a catalyst was found to be superior to the conventional inorganic acid method.

Titanium Dioxide Nanotubes Incorporated Bioactive Glass Nanocomposites: Synthesis, Characterization, Bioactivity Evaluation and Drug Loading

Nano bioactive glasses are known as suitable alternatives to repair the damaged bone tissues. In this research, novel sol-gel derived bioactive glass composites were synthesized through a reduction in the common weight percent of SiO2 substituted by 15 wt% of titanium dioxide nanotubes (TNTs) at two different steps by the synthetic procedure. The morphology, crystalline structure, and functional groups of the composites were evaluated through scanning electron microscopy (SEM), X-ray diffraction (XRD) and Fourier transform infrared (FTIR) analyses. Based on the SEM images, the step in which TNTs were added to the solution completely changed the morphology of the composite. Bioactivity tests were carried out by soaking the samples in the simulated body fluid (SBF) at the intervals ­of 14 and 28 days followed by the investigation of hydroxyapatite (HA) layer formation on the surface of the samples. According to XRD peaks at 2-theta angle of around 31 and 40 degrees, it was found that the presence of titanium dioxide nanotubes improved bioactivity after 14 days of immersion and both 58S-TNT composites were more bioactive than 58S bioglass, while 58S bioactive glass possessed more intense peaks of HA after 28 days of immersion in SBF. Furthermore, the drug loading characteristic of the prepared composites was examined and the results showed that the addition of nanotubes improved the drug loading performance of bioactive composites containing TNTs up to 70% compared to the 58S bioglass with 37% drug loading.

Atomistic insights into the structure and elasticity of densified 45S5 bioactive glasses.

Glasses have applications in regenerative medicine due to their bioactivity, enabling interactions with hard and soft tissues. Soda-lime phosphosilicate glasses, such as 45S5, represent a model system of bioactive glasses. Regardless of their importance as bioactive materials, the relationship between the structure, density, and cooling process has not been studied in detail. This hinders further development of glasses as biomaterials. We used molecular dynamics simulations to study the elastic and structural properties of densified 45S5 bioactive glass and liquids over a wide range of densities. We performed a systematic analysis of the glass structure to density relationship to correlate the change in the properties with the structural change to enhance the mechanical properties of bioactive glasses while preserving their bioactive nature. The results show that the glass structure tends to be repolymerized, as indicated by increased network connectivity and a tetrahedral to octahedral polyhedral transition. We were able to tailor the elastic properties while keeping the bioactivity of the glass. The results presented here will provide some guidance to develop bioactive glasses with enhanced mechanical properties.

Synthesis of 45S5 bioactive glass-ceramic using the sol-gel method, catalyzed by low concentration acetic acid extracted from homemade vinegar

In this paper, two different type of 45S5 bioactive glass-ceramic were compared. Both of them were prepared using the sol-gel method, with two different catalysts: 2 M nitric acid (NACBG) and 10 mM vinegar. Homemade vinegar (HMV) extracted from apples which contained up to 5% acetic acid, as an affordable and low concentration catalyst, was used for the synthesis of 45S5 bioactive glass ceramic called Homemade Vinegar Catalyzed Bioactive Glass (HMVCBG). The substitution of 2 M nitric acid by 87 mM acetic acid strongly reduces the concentration of the acid solution necessary for being a catalyst. The effect of these two catalysts on the structure, morphology, and properties of the bioactive glass-ceramics were investigated. Scanning electron microscopy (SEM) with energy dispersive X-ray spectroscopy (EDXS) and Brunauer–Emmett–Teller (BET) were used to assess surface characteristics and specific surface area, which showed more uniformity and more specific surface area in HMVCBG. Crystallinity and composition were analyzed by X-ray diffraction (XRD), X-ray fluorescence (XRF) and Furrier transform infrared spectroscopy (FTIR) which showed some crystalline phases in BG. In-vitro bioactivity was studied in Phosphate Buffer Saline (PBS) and SEM results showed that HMVCBG has a more uniform hydroxyapatite layer and formation occurs more quickly. This due to the fact that hydroxyapatite is dissolved at a lower rate at higher pH values and it takes longer to form at a higher pH. HMV can form more numerous porosities and a more specific surface area because of higher pH of acetic acid. Hence, PBS solution penetrates to the depth of HMVCBG and hydroxyapatite nucleation form in more spots in the depth of HMVCBG. Thus, HMVCBG has a higher bioactivity level than NACBG.

Preparation and characterization of 58S bioactive glass based scaffold with Kaempferol-containing Zein coating for bone tissue engineering.

The aim of this study was to prepare a porous scaffold out of 58S bioactive glass as the bare and coated with Zein to improve mechanical properties and acting as a carrier for Kaempferol controlled delivery. Porosity and morphology, mechanical properties, drug release behavior, bioactivity, cell attachment, and biodegradation of the scaffolds were evaluated accordingly. Obtained results indicated that the scaffolds coated by (7wt/v %) Zein solution, showed the highest mechanical strength (3.06 ± 0.4MPa) and desirable porous morphology. These scaffolds could support bioactivity, cell attachment, and provide sustained drug release in the safe range of Kaempferol concentration confirmed via 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide analysis. Overall, this study showed that the Zein-coated scaffold possesses superior properties rather than bare scaffold, and the scaffolds coated with 7wt/v % Zein solution could be considered as appropriate scaffolds for bone regeneration.

A New Generation of Electrospun Fibers Containing Bioactive Glass Particles for Wound Healing.

Chitosan fibers blended with polyethylene oxide (CHIT_PEO) and crosslinked with genipin were fabricated by electrospinning technique. Subsequently, CHIT_PEO bioactive glass composite electrospun mats were fabricated with the aim to achieve flexible structures with adequate mechanical properties and improved biological performance respect to CHIT_PEO fibers, for potential applications in wound healing. Three different compositions of bioactive glasses (BG) were selected and investigated: 45S5 BG, a Sr and Mg containing bioactive glass (BGMS10) and a Zn-containing bioactive glass (BGMS_2Zn). Particulate BGs (particles size < 20 μm) were separately added to the starting CHIT_PEO solution before electrospinning. The two recently developed bioactive glasses (BGMS10 and BGMS_2Zn) showed very promising biological properties in terms of bioactivity and cellular viability; thus, such compositions were added for the first time to CHIT_PEO solution to fabricate composite electrospun mats. The incorporation of bioactive glass particles and their distribution into CHIT_PEO fibers were assessed by SEM and FTIR analyses. Furthermore, CHIT_PEO composite electrospun mats showed improved mechanical properties in terms of Young’s Modulus compared to neat CHIT_PEO fibers; on the contrary, the values of tensile strain at break (%) were comparable. Biological performance in terms of cellular viability was investigated by means of WST-8 assay and CHIT_PEO composite electrospun mats showed cytocompatibility and the desired cellular viability.

Influence of particulate alkaline biomaterial remnants in dentin on the adhesion of two resin-based bonding systems.

To study aimed to evaluate the influence of infiltrated alkaline biomaterials on the bond strength of composite obtained with etch and rinse and universal adhesive systems. Coronal dentin of extracted human molars was exposed and infiltrated with calcium hydroxide, MTA, or bioactive glass 45S5 powder. Non-infiltrated dentin specimens served as controls. Subsequently, dentin was either acid-etched or not, and remaining alkali content was measured by pH induction in water. Shear bond strength of an etch-and rinse system (Adper Single Bond 2) on etched dentin was then compared to that of a universal counterpart (Single Bond [(Scotchbond) Universal] by the same manufacturer. Data was compared between groups using one-way ANOVA and Tukey's HSD. On sound dentin, the etch-and-rinse system adhered stronger than the universal system (p < .05). All particles rendered the dentin alkaline. Acid etching reduced the alkali content in the infiltrated dentin. However, bond strength values were stronger with the universal system without etching than with the etch-and rinse counterpart, which lost more than 60% of its adhesiveness. The universal adhesive system was less susceptible to alkaline contamination of the dentin than the etch-and-rinse counterpart. Based on the similar composition of the two systems under investigation, this is likely to the due to its 10-MDP component.

Effect of feedstock and plasma gun on the microstructure and bioactivity of plasma sprayed bioactive glass coatings

Plasma-sprayed 45S5 bioactive glass coatings were manufactured using two types of powder feedstocks (commercial and lab-made 45S5 glass) and two plasma torches (single and triple cathode) to analyse their influence on the microstructure and bioactivity of the coatings. Besides, the volatilisation of glass oxides such as Na2O and P2O5 during the deposition was studied. All coatings were microstructurally characterised by scanning electron microscopy, X-ray diffraction and X-ray dispersive energy analysis. Moreover, the bioactivity of the sprayed coatings was studied by immersing the coatings in Simulated Body Fluid until 14 days.The coatings obtained using the triple cathode torch showed similar thickness, less total porosity and greater microstructural homogeneity than the coating deposited with the single cathode torch. X-ray dispersive energy analysis revealed lower amounts of sodium and phosphorus at the surrounding of the lamellae, due to its volatilisation during the formation of the coatings. The volatilisation of these elements varied depending on the type of feedstock and plasma jet enthalpy. Concerning the bioactivity of the coatings, all of them have developed a hydroxycarbonate apatite layer on their surface.

An experimental and theoretical approach on stability towards hydrolysis of triethyl phosphate and its effects on the microstructure of sol-gel-derived bioactive silicate glass

The sol-gel method is versatile and one of the well-established synthetic approaches for preparing bioactive glass with improved microstructure. In a successful approach, alkoxide precursors undergo rapid hydrolysis, followed by immediate condensation leading to the formation of three-dimensional gels. On the other hand, a slow kinetics rate for hydrolysis of one or more alkoxide precursors generates a mismatch in the progression of the consecutive reactions of the sol-gel process, which makes it difficult to form homogeneous multicomponent glass products. The amorphous phase separation (APS) into the gel is thermodynamically unstable and tends to transform into a crystalline form during the calcination step of xerogel. In the present study, we report a combined experimental and theoretical method to investigate the stability towards hydrolysis of triethyl phosphate (TEP) and its effects on the mechanism leading to phase separation in 58S bioactive glass obtained via sol-gel route. A multitechnical approach for the experimental characterization combined with calculations of functional density theory (DFT) suggest that TEP should not undergo hydrolysis by water under acidic conditions during the formation of the sol or even in the gel phase. The activation energy barrier (ΔG‡) showed a height of about 20 kcal·mol−1 for the three stages of hydrolysis and the reaction rates calculated for each stage of TEP hydrolysis were kFHR = 7.0 × 10−3s−1, kSHR = 6.8 × 10−3s−1 and kTHR = 3.5 × 10−3s−1. These results show that TEP remains in the non-hydrolyzed form segregated within the xerogel matrix until its thermal decomposition in the calcination step, when P species preferentially associate with calcium ions (labile species) and other phosphate groups present nearby, forming crystalline domains of calcium pyrophosphates permeated by the silica-rich glass matrix. Together, our data expand the knowledge about the synthesis by the sol-gel method of bioactive glass and establishes a mechanism that explains the role played by the precursor source of phosphorus (TEP) in the phase separation, an event commonly observed for these biomaterials.

Preparation, characterization and in vitro biological response of simultaneous co-substitution of Zr+4/Sr+2 58S bioactive glass powder

In the present study, structure of zirconium-containing bioactive glass (58S-BG (Zr-BG)) with optimal fixed Zr content (5 mol.%) was modified by incorporation of strontium (Sr). These Zr and Sr-containing BGs (ZS-BGs) were synthesized by sol-gel method and substitution of Ca with modifier ions (Sr content = 0, 3, 6, 9, and 12 mol.%). The results obtained from characterization by X-ray diffraction (XRD), scanning electron microscopy (SEM), Fourier-transform infrared (FTIR), and energy-dispersive X-ray spectroscopy (EDS) techniques from surface of all the ZS-BGs revealed formation of hydroxyapatite (HA) after 7 days of immersion in the simulated body fluid (SBF) solution. Evaluation of changes in the SBF solution, by monitoring pH variations and ions҆ concentration, was in agreement with the results of morphological and structural investigations. The in-vitro biological function of synthesized BGs was studied through (MTT) assay and alkaline phosphatase (ALP) activity analysis. The results showed that all the specimens significantly stimulated proliferation and viability of MC3T3 osteoblast-like cells. Furthermore, antibacterial studies confirmed less resistance of methicillin-resistant Staphylococcus aureus (MRSA) bacteria against ZS-BGs. Eventually, the results of in-vitro bio-analysis were clarified and confirmed by two cell staining techniques of Live/Dead and Dapi/Actin. This confirmation was achieved by observing the increased quantity of live cells and their nuclei as well as the decreased number of dead cells after co-culturing with all ZS-BGs.

On the role of alginate coating on the mechanical and biological properties of 58S bioactive glass scaffolds

In this study, novel 3D porous alginate-coated 58S bioactive glass scaffolds were fabricated through a foam replication method using a combination of amorphous 58S bioactive glass structure and sodium alginate. The formation of the alginate coating on the surface of the struts of BG scaffolds was confirmed. The effect of alginate coating on the microstructure, mechanical properties, biodegradability, biomineralization, adhesion, viability, and differentiation of human bone marrow-derived mesenchymal stem cells (hMSCs) on the 58S BG scaffolds were evaluated. A 45.2% increase in hMSC's viability and a 3.4-fold increase in ALP activity of the 1AlBG scaffold in the absence of an osteogenic differentiation media compared to an uncoated BG scaffold were observed. Notably, gene expression analysis exhibited that the 1AlBG scaffold resulted in accelerated osteogenic differentiation of hMSCs, as expression of COL-1, RUNX2, and OCN increased after 14 days. Results revealed a significant increase of antibacterial inhibition in the 1AlBG scaffold in comparison to the BG scaffold. Based on the microstructural, mechanical, and biological investigations, the 1AlBG scaffold exhibited enhanced mechanical and biological properties, making it a promising candidate for bone regeneration. Overall, our findings have highlighted the potential of alginate-coated BG scaffolds to stimulate bone regeneration through stem cell osteoinduction.

New sol-gel-derived magnetic bioactive glass-ceramics containing superparamagnetic hematite nanocrystals for hyperthermia application

Although the three main phases of iron oxide – hematite, maghemite, and magnetite – exhibit superparamagnetic properties at the nanoscale, only maghemite and magnetite phases have been explored in magnetic bioactive glass-ceramics aimed at applications in cancer treatment by hyperthermia. In this work, it is reported for the first time the superparamagnetic properties of hematite nanocrystals grown in a 58S bioactive glass matrix derived from sol-gel synthesis. The glass-ceramics are based on the (100-x)(58SiO2-33CaO-9P2O5)-xFe2O3 system (x = 10, 20 and 30 wt%). A thermal treatment leads to the growth of hematite (α-Fe2O3) nanocrystals, conferring superparamagnetic properties to the glass-ceramics, which is enough to produce heat under an external alternating magnetic field. Besides, the crystallization does not inhibit materials bioactivity, evidenced by the formation of calcium phosphate onto the glass-ceramic surface upon soaking in simulated body fluid. Moreover, their cytotoxicity is similar to other magnetic bioactive glass-ceramics reported in the literature. Finally, these results suggest that hematite nanocrystals' superparamagnetic properties may be explored in multifunctional glass-ceramics applied in bone cancer treatment by hyperthermia allied to bone regeneration.

Adhesion improvement and in vitro characterisation of 45S5 bioactive glass coatings obtained by atmospheric plasma spraying

Plasma sprayed bioactive glass coatings were studied using crushed 45S5 bioactive glass powder. It is widely accepted that plasma sprayed coating microstructure is highly affected by the characteristics of the powder and the parameters set on the spraying process. Once the coating deposition was optimized, two strategies were carried out to analyse their effect on the coating adhesion: cooling with carbon dioxide while spraying and a post heat treatment to the as-sprayed coatings.Scanning electron microscopy and X-ray diffraction were used for analyse the obtained coatings. Additionally, coating adhesion to the substrate and degradation of the coatings in Tris buffer solution were evaluated for the different samples studied.Coatings have been tested in vitro to evaluate their response by immersion in simulated body fluid, Hank's Balanced Salt Solution.The results show an increase in the adhesion strength for the heat treated samples due to the stress relaxation achieved above glass transition temperature. Moreover, in the bioactivity test an apatite layer at the coatings surface was produced for all the strategies studied.

Effect of hydroxyapatite and 45S5 bioactive glass addition on a dental adhesive resin cement

AbstractObjectiveThis study evaluates the mechanical, physical, and biological properties of resin‐based dental cement (RelyX ARC, 3M ESPE) incorporated with hydroxyapatite and 45S5 bioactive glass.MethodsBioactive glass (45S5) and hydroxyapatite (HA) were added to cement in different proportions (10 wt% and 20 wt%). Three‐point flexural strength, microhardness, surface roughness, film thickness, contact angle, and color were evaluated. Cytotoxicity in human osteoblasts culture (MG63) was evaluated. Statistical analysis was performed using one‐way and two‐way ANOVA and Tukey tests (α &lt; 0.05).ResultsThe addition of HA and 45S5 bioactive glass reduced the flexural strength of the composite. However, the resistance value was greater than the minimum values of the ISO standard. Film thickness was below the maximum values, except for the 20% HA. The contact angle was constant at 24 hours and 14 days, except for 10% 45S5 group, which presented a significant decrease in mean contact angle. Cellular viability for all groups increased from the first to the 14th day. Additionally, the cellular viability was above 50%.ConclusionsIn vitro incorporation of 45S5 bioactive glass on resin cement resulted in a promising luting dental material with adequate mechanical properties, thin film thickness, improved wettability, and adequate biocompatibility.

Improvement of the mechanical and biological properties of bioactive glasses by the addition of zirconium oxide (ZrO2 ) as a synthetic bone graft substitute.

In this study, mechanical properties of bioactive glass (BAG) synthetic bone graft substitute was improved by containing ZrO2 (ZrO2 -BAG), while maintaining advantageous biological properties of BAG such as osteoinductive and osteoconductive properties. The ZrO2 -BAG was produced by adding ZrO2 in the following proportions to replace Na2 O in 45S5 BAG: 1% (Zr1-BAG), 3% (Zr3-BAG), 6% (Zr6-BAG), and 12% (Zr12-BAG). Properties including XRD, XPS, SEM, DSC, fracture toughness, and Vickers microhardness were evaluated. To assess the biological properties, Ca/P apatite formation, ion release, degradation rate, cell proliferation, ALP activity (ALP), and alizarin red S staining assay (ARS) were evaluated. Also, expression of osteogenic differentiation markers, Osteopontin (OPN), confirmed by immunofluorescence staining. Finally, an in vivo test was carried out to by implanting ZrO2 -BAG into the subcutaneous tissue of rats. The results of each test were statistically analyzed with one-way ANOVA followed by Tukey's post hoc statistical test. Amorphous ZrO2 -BAG was successfully produced with increased mechanical properties as the ZrO2 content was increased. Additionally, ZrO2 -BAG exhibited a slower ion release and degradation rate compare to BAG without ZrO2 . Bioactivity of ZrO2 -BAG was confirmed with apatite layer formed on the surface, significantly higher proliferation rate and significantly enhanced ALP and the degree of ARS of the cells compare to respective controls. The tissue reactions observed in the in vivo study showed neo-formed vessels after implantation of ZrO2 -BAG.

Sol-gel-derived 58S bioactive glass containing holmium aiming brachytherapy applications: A dissolution, bioactivity, and cytotoxicity study

Bioactive glasses containing rare earth elements have been proposed as promising candidates for applications in brachytherapy of bone cancer. However, their safety relies on a proper dissolution to avoid radioactive materials in the human body, and desirable bioactive properties to regenerate the bone defect caused by the tumor. In this work, we proposed a new series of sol-gel-derived bioactive glasses containing holmium oxide, based on the system (100-x)(58SiO2-33CaO-9P2O5)-xHo2O3 (x = 1.25, 2.5 and 5 wt%). The glasses were characterized regarding their dissolution behavior, bioactivity, and cytotoxicity with pre-osteoblastic cells. Also, in the dissolution experiments, the Arrhenius and Eyring equations were used to obtain some thermodynamic properties of glass dissolution. The results evidenced that the addition of holmium ions in the glass structure decreased the energy barrier of hydrolysis reactions, which favors glass dissolution in an early-stage. However, in the long-term, the strength of Si-O-Ho bonds may be the cause of more stable dissolution. Besides, glasses containing holmium were as bioactive as the 58S bioactive glasses, a highly bioactive composition. Cytotoxicity results showed that all glasses were not cytotoxic, and the composition containing 5 wt.% of Ho2O3 enhanced cell viability. Finally, these results suggest that these glasses are suitable materials for brachytherapy applications due to their proper dissolution behavior, high bioactivity, and high cell viability.

Influence of the porosity and type of bioglass on the micro-mechanical and bioactive behavior of coated porous titanium substrates

In this study, the best equilibrium among adherence, micro-mechanical properties and coating bioactivity of bioactive glasses (45S5 and 1393) on porous titanium substrates has been explored and their potential uses for bone tissue implants. Porous titanium discs with different porosities (30 and 60 vol.%) and pore size distributions (100–200 and 355–500 µm) were utilized to rationalize their influence on both properties and performance. It was corroborated that porous samples produced a reduction in micro-hardness (~ 2000–4000 N/mm2) and the elastic modulus (~25–50 GPa), obtaining values closer to those of human bones, as well as induced a beneficial role to integrate the coatings. On one hand, bioglass 1393 presented greater capacity for pore infiltration while 45S5 was more bioactive. The results explained the better adherence and microhardness for bioglass 1393 and the significant formation of hydroxyapatite and calcium phosphates of bioglass 45S5, confirmed by 29Si MAS NMR.

Investigation of 58S bioactive glass tablets

In this study, bioactive glass powders were successfully synthesized by using the sol-gel process and bioactive glass powders were tabletted by direct dry pressing method. The morphology and surface properties of bioactive glass tablets were examined via field emission scanning electron microscope (FE-SEM) devices. X-ray diffraction (XRD) was utilized to evaluate the phases formed in the sol-gel bioactive glass tablets. Surface characterization of the tablets immersed in simulated body fluid (SBF) was carried out with XRD, FE-SEM, and Fourier transform infrared (FTIR). XRD, FTIR, and EDS analysis proved that the sample contained hydroxyapatite. Also, the in vitro mineralization assay demonstrated that bioactive glass tablets are capable of inducing the creation of hydroxyapatite after dipped in SBF. All analyze results showed that bioactive glass tablets have good apatite-forming activity.

Mechanical Properties of Bioactive Glass Fabricated Using Natural Resources Materials

Bioactive glass use silica (SiO2), calcium carbonate (CaCO3), sodium carbonate (Na2CO3), phosphorus pentoxide (P2O5) as raw materials. In this work, bioactive glass (BG); 45S5 bioactive glass was synthesized using natural resources materials; rice husk ash (RHA) as silica (SiO2) source and seashell (SS) as calcium carbonate (CaCO3) source through melt derived method. All raw materials were melted at 1400 °C and water quenched. The glass frit obtained was milled and sieved then analyzed using X-ray diffraction (XRD), Fourier Transform Infrared spectroscope (FTIR) and Scanning Electron Microscope (SEM). The mechanical properties 45S5 BG pellet was observed through diametral tensile stress (DTS). The XRD and FTIR pattern for all sample synthesized using natural resources raw materials show similar pattern with control sample 45S5 synthesis using pure raw materials. The mechanical properties for all samples also have not significantly different with control samples

Polymerization shrinkage behaviour of resin composites functionalized with unsilanized bioactive glass fillers

Previous work has shown that partial replacement of reinforcing fillers with unsilanized silica particles can diminish polymerization shrinkage stress of dental resin composites. The aim of the present study was to investigate whether such an effect can be attained by using unsilanized bioactive glass (BG). Incorporating BG fillers into resin composites is interesting due to their potential for exerting caries-preventive effects. Experimental light-curable composites with a total filler load of 77 wt% were prepared. Reinforcing fillers were partially replaced with 0–60 wt% of BG 45S5 and an experimental low-sodium fluoride-containing BG. The following properties were investigated: linear shrinkage, degree of conversion, shrinkage stress, maximum shrinkage stress rate, and time to achieve maximum shrinkage stress rate. The diminishing effect of BG 45S5 on shrinkage stress was mediated by a decrease in degree of conversion caused by this BG type. In contrast, as the degree of conversion remained unaffected by the experimental BG, the resulting shrinkage behaviour was governed by the effect of varying amounts of silanized and unsilanized fillers on material’s viscoelastic properties. The replacement of silanized reinforcing fillers with unsilanized BG did not reduce polymerization shrinkage stress unless the reduction was attained indirectly through a diminished degree of conversion.