Percent composition of agriresidue derived materials (Traces: Ti, Cr, Ni, Ga, Br, Rb, Mn, Sr, Cu).
The transformation of subproducts and residues is of utmost importance, especially when it closes an industrial cycle, where solutions for environmental problems such as contamination leads towards a sustainable development, converting residues into value added products. Materials prepared from agricultural residues may be considered as Renewable Raw Materials [1,2]. Using these materials as a source for useful materials avoids the expense of employing other materials that are often non-renewable. This philosophy is denoted as “cradle to grave”, since the residues of a company are used by the same or others .
Agriculture is one of the pillars of society, especially given the increasing world population. Countries which have large agricultural resources, such as Spain, also produce vast amounts of residues and wastes, that can be an environmental hazard, difficult to store, easily generating leakages and greenhouse gases such as ammonia, methane and carbon, nitrogen and sulphur oxides when burnt, therefore negatively impacting on the environment. However, these agriwastes can also be considered as a source of low cost renewable raw materials (RRM) that with the proper treatment can have applications in a wide range of processes,
The work presented here uses agricultural wastes to prepare materials for effluent cleaning with rice and beer production wastes, etherification of glycerol from biodiesel production, enzyme immobilisation and use in biodiesel preparation and Bioecomaterials for tissue engineering and controlled desorption of bioactive substances.
2. Effluent cleaning with materials derived from rice and beer production wastes
Industrial effluents often have high flows with variable concentrations of toxic substances, where adsorption is an ideal process for their decontamination due to its good economic turnover and the possibility of recovering the contaminants by desorption. The process of adsorption is based on the diffusion of sorbates to the adsorbent surface, followed by the inclusion inside the pore structure where they are stored. Here the textural properties of the adsorbents (surface area and porosity) are of the utmost importance .
Distilleries Muñoz Galvez (DMG) is a leading Spanish Company that manufactures and exports Essential Oils and Aromatic Raw Materials (Fine Chemicals) as well as Fragrances and Flavours worldwide . The challenge faced by the research group (CDTI project)  was to clean DMG wastewaters that contained terpenes as the main residue, as expected given their origin, thus reducing water consumption, economic expenditure and possible environmental hazards. Aiming to decrease the economic costs, and increase the project´s environmental approach, agriresidues from beer and rice production were used to prepare materials capable of wastewater decontamination that were compared to commercial adsorbents. Previous knowledge of the researchers was applied and optimised to the wastewater composition and treatments [8,9]. The results indicated better adsorption and therefore cleaning capacities in the residue derived materials than in the commercial ones. The chemical oxygen demands after wastewater treatment were low enough for the treated water to be discharged in accordance with the legal requirements.
The agriresidues chosen for this study were beer bagasse and rice husk. Beer bagasse, from Mahou San Miguel (Spain)  (designated as BBM), is a residue from beer production that has
Rice husk (RH) from DACSA (Spain)  is an agriresidue from rice production, of difficult storage and transport due to its high volume to weight ratio. On calcining this residue, a material with more than 97% silica is produced, that has also low amounts of calcium and potassium [13-15].
The agriresidues utilized as raw materials in this study were used as received, in the case of rice husk, or after a drying step at 150°C in the case of the BBM, to inhibit further fermentation due to its high water content. The RH and the dried BBM were analysed by TG-DTA in air to determine their thermal stabilities and design the material preparation. Details of experimental set up can be found in . Figure 1 shows these data.
From these results the agriresidues loose up to 90 (BBM) and 87 (RH) percent weight, when calcined up to 800°C, where the weight losses were an endotherm due to water loss up to 200 °C and two exotherms at 340 °C and 520 °C (BBM) or 340 °C and 470 °C (RH) due to decomposition/transformation of the organic matter to volatile organic substances and carbonization .
After studying the reproducibility of the materials the temperature chosen to prepare the adsorbents through thermal treatment of the agriresidues was 350 °C for 2, 4 or 6 h. These materials were designated as BBM2, BBM4, BBM6, RH2, RH4 and RH6. The composition analyses of these agriresidue derived materials are included in Table 1. From these results it was clear that under these conditions the materials derived from BBM were mainly carbonaceous whilst those derived from RH have siliceous structure and the content of the elements that form volatile species (CO2, H2O, NOX, oxychlorides,
Textural analyses of the agriresidue derived materials were carried out by mercury intrusion porosimetry (MIP) and the data obtained are included in Figure 2 and Table 2. Description by the authors of the experimental analyses for MIP can be found in reference 16.
In this technique, the pores below 300 nm correspond to those inside the particles, while those at higher values are due to interparticle voids. As can be seen in all the materials prepared, the curves coincide for values below 1000 nm. As expected, in general, there was an increase in the total pore volume on increasing the calcination time, due to decomposition of volatile compounds, producing extra porosity, in agreement with TG-DTA analyses.
From this data it can be observed that the porosity, surface area and medium pore size increase with time of calcination and the surface areas were lower but the pore sizes higher for the residue derived materials compared to the commercial activated carbon.
The analysis of DMG wastewater was carried out by GCMS (Figure 3), the experimental details for this technique can be found in reference 17. From these analyses the presence of
The DMG wastewater treatments were carried out by using 0.5 g of carbon (commercial or Ecomaterial), and 30 mL of the DMG wastewater (to ensure reproducibility of the measurements), these slurries were magnetically stirred and 2 mL aliquots of the original and treated wastewaters were extracted at increasing times with the same volume of di-isopropylether, dried over sodium sulphate and analysed by GCMS (Figures 4 and 5).
These results indicated that the carbon prepared from beer residues had better adsorption capacities for cleaning the DMG wastewater than the commercial one, especially with regards to the high molecular weight substances. This can be related to the different textures, since the commercial carbon has smaller pores that can not easily accommodate the high molecular weight substances. The chemical oxygen demand (COD) of the original and treated wastewaters was studied according to the Spanish UNE 77004, equivalent to ISO 6060:1989:
where C: concentration of Fe(II) sulphate and ammoniumin mol/L, V0: volume in mL before dilution, V1: volume in mL of Fe(II) sulphate and ammonium solution for blank analysis, V2: is the volume in mL of Fe(II) sulphate and ammonium solution for assay, 8000 is molar mass in mg/L of ½ O2. The COD results are in mg O2/L. The value of the method has been checked with a 0.425 g of potassium hydrogenphtalate (KC8H5O4), dried at 105°C, diluted in 1000 mL distilled water with a COD standard value of 500 mg O2/L (+/- 20). Variabilities in COD analyses were less than 2 % . The results obtained for the COD reduction of the wastewater, with the different materials are included in Table 3 (Percent reduction of COD after room temperature wastewater treatment with adsorbents until constant COD (usually
The high effectiveness of the residue derived materials compared with the commercial carbon should be noted. The material with greater cleaning ability was RH6 (73% reduction), which allowed a water with COD of 960 mg O2 /L. Comparing the textural data with COD determinations, it can be said that there was a direct correlation between the pore diameter of the solids and their COD reduction capacity. Thus, wastewater treatment with residue derived materials has been shown to be an economical and environmentally sound process that should be further developed .
3. Etherification of glycerol from biodiesel production with company´s own residues
The need for renewable energies in general and biodiesel in particular, indicates that optimising the production process is of vital importance. Biodiesel production generates
This research was undertaken with the aim to use Acesur-Tarancon´s own subproducts to prepare catalysts to transform the company´s glycerol (from their biodiesel plant) to diglicerol ethers. The catalysts prepared in this way are in fact Ecomaterials and their origin makes them competitive with commercial ones. Production of ethers with more than three glycerol molecules competes with diethers and thus control of the selectitivy is important, especially keeping in mind that the diglycerol ethers (Figure 6) are produced at short reaction times, which gives the process an added value [20, 22].
Etherification of glycerol with acid catalysts was found to be difficult to control. However, the catalytic transformation of glycerol into ethers, carried out with basic catalysts allows more controllable results. Furthermore, the use of heterogeneous catalysts
The oligoglycerols are gaining more and more interest as products used in cosmetics, food-additives or lubricants . Short overviews about the synthesis of glycerol oligomers from di- to pentaglycerol have been published by Rollin
The conditions used for glycerol etherification were chosen with basic catalysis, since the sunflower oil production agriresidue derived materials (RP45), given their composition of alkaline (26 % potassium) and alkaline-earth cations (5 % magnesium and 7 % calcium) are basic in nature. TGMS of adsorbed acetic acid indicated that RP45 contained basic centres of low (100-200 °C), medium (200-500 °C) and high basicity (550-650 °C) (Figure 7) (see procedure for basicity measurement in reference 14) and can catalyse Knoevenagel condensation reactions .
The reactions were carried out in abscense of solvent, under inert atmosphere to limit overoxidation, controlling temperature and time of reaction to optimise the economics [30, 31]. The work described here was carried out to produce diglycerol ethers in the absence of solvent, under inert atmosphere to avoid overoxidation, using catalysts derived from sunflower oil production residues, which have medium and high basic strengths. The analysis of reaction products was carried out by GC-MS (conditions as in reference 14).
Optimisation of the reaction conditions (Figure 8) showed that with 240°C, under inert atmosphere (nitrogen flow) and a ratio catalyst/glycerol = 1/50, after 4 h the conversions of glycerol and selectivities to diglycerols were optimum. At lower temperatures the conversions were low and with higher temperatures the selectivity to diglycerols decreased due mainly to uncontrolled formation of polyglycerols and oxidised compounds (mainly glycolic and glyceric acids).
Main compounds found in the reaction carried out in this work are summarised in Figure 9.
The catalyst chosen for reference was sodium hydroxide, which was dissolved in the glycerol, where from the bibliography the amount of NaOH was chosen to give a molar ratio NaOH/Glycerol = 50 . The homogeneous reaction with diluted NaOH (1 g/50 mls glycerol, 240 °C), reached 20 % conversion with glycerol ethers mainly cyclic (18-20 min analysis) after 3 h of reaction, lower temperatures gave very low conversions and higher temperatures or times decreases the selectitivty to diglycerol ethers due to unwanted triglycerol compounds (Figure 10). The reaction with RP45 (1 g/50 mls glycerol) led to a ratio between linear/cyclic diglicerol of 1/4, while on using homogeneous reaction, only cyclic diglyerol was produced under the conditions used (Figure 9). On increasing the NaOH/Glycerol or RP45/glycerol ratios the selectivity decreased due to unwanted glyceric and glycolic acids due to over oxidation and triglycerols .
Given the basicity of RP45 (see Figure 7) it carbontaes easily in the open atmosphere due to reaction with CO2, therefore it was important to activate this material
4. Lipases immobilised on materials prepared with agriresidues derived from rice production.
Lipases are enzymes of the hydrolases family, with capabilities such as hydrolysing triglycerides to diglycerides, monoglycerides, fatty acids and glycerol, by reaction of the carboxylic ester bonds. More than 25 % of enzymes used in biotransformations are lipases. However, their high production cost are their main disadvantage for industrial uses like soap and detergent production, baby milk preparation, hydrolysing the grease in milk or production of pharmacological substances. In the human body these substances are important since they facilitate fats adsorption.
Lipase immobilisation is of interest since it allows their reuse and increases their resistance to inactivation. For industrial applications, several properties are important
The thermal stabilities of the residues in air were analysed by thermal techniques (TGA–DTA) on a Netzsch 409 EP Simultaneous Thermal Analysis device. Approximately 20–30 mg of powdered samples were heated in an air stream of 75 mLmin−1 at a heating rate of 5 °Cmin−1 from ambient to
The TXRF analysis of the materials derived from heat treated rice husk (RH47, RH45, RH26) indicated that they contain
The crystallinity of the materials was recorded by X-ray diffraction (XRD) on a Seifert 3000P diffractometer, using Cu Kα1 radiation:
Fourier transformed infrared transmission spectra (FTIR) of materials obtained on a Nicolet 40 FTIR spectrophotometer in the wavenumber range of 4000–400 cm−1, using a 1/100 dilution in KBr indicated the presence of bands at 900-1200 cm-1 and at 400-600 cm-1, corresponding to metal-oxygen bonds (800 cm-1 is O-Si-O symmetric stretching vibrations), given the oxide structure of the materials freshly calcined and bands of OH- at
The specific surface areas measured by N2 adsorption at 77 K after outgassing overnight at 150 °C and employing the BET method for data analyses in a Sorptomatic 1800 instrument (Table 4) indicated that RH derived materials are mesoporous with type IV isotherms and wide pore size distributions, and RP derived materails are non-porous with type II isotherms and non-existent hysteresis loops. Specific surface areas are listed below.
The porosities in pores from 300 μm down to 7.5 nm were determined by mercury intrusion porosimetry (MIP) using CE Instruments Pascal 140/240 porosimeter on samples previously dried overnight at 150 °C, the Washburn equation was employed, assuming a non-intersecting cylindrical pore model and the recommended values for the mercury contact angle and surface tension of 141 ° and 484 mNm−1, respectively. These studies show that materials RH26 and RP47 have pore volumes, mesopores areas, medium pore radii and medium particle sizes as follows: RH26 (0.08 cm3g-1, 27 m2g-1, 80 µm and 90 µm), RP47 (0.02 cm3g-1, 4 m2g-1, 15 µm and 30 µm).
In order to measure the basicity of the solids acetic acid was previously adsorbed onto the powder materials and subsequently the mas of 44 was recorded against temperature with a quadrupole mass spectrometer, M3 QMS200 Thermostar coupled to Stanton STA model 781 TG/DTA apparatus. For these analyses approximately 50 mg of the materials were dosed with acetic acid, transferred to the crucible placed within the Stanton TG-MS, where they were subsequently flushed with nitrogen gas at room temperature in order to desorb any loosely bound physically adsorbed acetic acid, until a constant weight was attained. The decomposition of the chemisorbed acetic entities was then achieved by increasing the temperature under a nitrogen flow at a heating rate of 5 °Cmin-1.
The amount and temperature of evolution of the CO2 signal gave an indication of the strength and amount of basic sites. The CO2 signal was calibrated from the decomposition of a known amount of calcium oxalate. These measurements indicated the presence of basic groups of high (>500 °C), medium (300-500 °C) and low basicity (100-300 °C) for RP materials, as corresponds to their content in alkaline and alkaline-earth cations and the RH materials had lower amounts of basic centers and of lower strength than the RP materials (Figure 13) .
The immobilisation process and measurement of activity was undertaken until there was no significant variation in activity. Subsequently, the biocatalyst was filtered, washed, dried over P2O5 and the enzymatic activity was studied at 30 °C, using a Mettler Toledo (modelo DL-50) pH-stato at pH=8.0 with 0.1 N NaOH titrating agent. As reaction medium 19 mL of 1nM tris-HCl buffer at a pH 8.0, 0.6 mL of acetonitrile and 0.4 mL of tripropionine as reaction substrate. A blank test was done to measure spontaneous hydrolisis (without enzyme and only with the triglyceride and the reaction medum). This technique consists of the controlled addition of a basic solution to maintain the pH, being then the titration proportional to the production of acid and therefore to the reaction rate.
The enzyme immobilisation on RH materials indicated that during the initial hours the percentage of immobilised enzyme grows but after 24 h there was no more absorption, however, when RP materials were used there was a continuous increment of immobilised enzyme, probably due to their lower pore volumes compared to RH materials. The materials that immobilised more enzyme were RH45 and RH26, where the latter was chosen for further studies since it had the highest catalytic activity. For the enzymatic activites, lipase Rhizopus oryzae expressed on levadura Pichia pastori, from the Autonoma University of Barcelona, was used. The lipase received as a solid was used to prepare the enzymatic solution of 20 mg/ml with sodium phosphate buffer 100 mM and pH 6.5, incubated stirring for 1 h at 4 °C and centrifuged to eliminate any solid residue .
The measurements of enzymatic activity, in sobrenadantes, control and stock solutions were carried out in a plate reader Versamax, using 10 mM
The experiments to study the reactions of hydrolysis were carried out using different enzymes, the test reaction of biodiesel synthesis by transesterification of triglycerides (trilaurin or triolein, Figure 14) with metanol or etanol was done where the main products were ethyl or methyl oleate or laurate and as secondary products mono and diglycerides and the corresponding fatty acid due to triglyceride hydrolysis.
Transesterification reactions were carried out with 50 mM ester concentrations and triglyceride:alcohol molar ratio 1:4, using 2-methtl-2-butanol (2M2B) as solvent, 20 mg/ml of enzyme, 45 °C and 300 rpm stirring speed. The progress of reaction was quantified by means of TLC chromatography, using a solution of hexane, ethyl acetate and glacial acetic acid (90:10:1) and developed by a solution of etanol, water, glacial acetic acid and a dye (
Transesterification of triglycerides with methanol and ethanol produce mainly methyl and ethyl oleate and secondary compounds such as fatty acid esters (mono- and dilaurin, monoolein and diolein) and the corresponding fatty acid due to the triglyceride hydrolysis. On using Novozym 435, trilaurin disappears after 24 h of reaction, because when the trilaurin is consumed, ethanol interacts with the diglyceride. Regarding the use of Lipozyme TL-IM, ethyl laureate formation is slower, although trilaurin also gets consumed and the formation of acid is lower, because Lipozyme TL-IM has a lower amount of water than Novozym 435. In the case of Lipozyme RM-IM, the formation of ethyl laureate is not so important as in the cases stated before, and at 24 h there is still a lot of unreacted trilaurin.
Amongst the materials used, those derived from rice husk show higher capacities for lipase absorption. It should be noted that RH45 and RH26 are the materials that have highest surface areas, but taking into account the amount of enzyme absorbed, it is clear that not only the textural characteristics are defining the behaviour of the supports but the amount and strength of the basic sites was also important, much higher in the RP materials. RH26 was chosen for preparation of immobilised enzyme for biodiesel synthesis in comparison with commercial enzymes. The ROL enzyme supported on RH26 prepared in the group had a higher activity than Novozyme 435 and similar to that of Lipozyme TL-IM, both lipases widely used in biocatalysis. Trilaurin was consumed in
The biocatalysts prepared by immobilisation of the lipase on agriresidue derived materials, given their renewable origin and low cost, seem to be an attractive option for reducing costs and environmental impact of these processes [37, 38].
5.1. Tissue engineering (Dental and Bone replacement therapies)
Bone tissue engineering is one of the most promising approaches to be used as an alternative to conventional autogenic or allogenic surgical techniques for bone tissue repair . Bone grafts are used to stimulate the formation of new bone in many conditions such as congenital anomalies, cancers, and trauma or to improve the regeneration of bone tissue around surgically implanted devices.
An ideal bone graft or scaffold should be made of biomaterials that emulate the structure and properties of natural bone extracellular matrix providing all the necessary environmental cues present in natural bone. The tissue regeneration capacity of these bone grafts is measured in terms of their osteogenic, osteoconductive and osteoinductive potential. The osteogenic potential of a bone graft is given by cells involved in bone formation, such as mesenchymal stem cells, osteoblasts, and osteocytes. The term osteoconductive refers to the scaffold or matrix which stimulates bone cells to grow on its surface. Osteoinductive capacity of a bone graft is perhaps the most important property in bone healing as it refers to the stimulation of mesenchymal stem cells to differentiate into preosteoblasts to begin the bone-forming process .
A possible therapy for the treatment of skeletal defects has arisen with the use of synthetic materials as bone substitutes. Tissue engineering strategies based on the use of biocompatible and biodegradable porous materials that act as structural templates or scaffolds to guide the growth and development of new bone tissue, supporting both extracellular matrix formation and cell-cell interactions . Due to their similarity to the chemical composition of bone, calcium phosphates can be used to regenerate osteoporotic bone as coatings that improve orthopedic implants, and for odontostomatologic applications, where their particle and crystal sizes are important parameters to be controlled to optimise these processes . Calcium phosphate-based scaffolds exhibit osteoconductivity, bioactivity and resorbability
Biomaterials based on composites of calcium phosphates and silica have the ability to bond directly to bone and thus enhance bone formation through supply of chemicals needed to support cell function and tissue formation. Furthermore, it has been found that addition of silica to the calcium phosphate scaffolds was beneficial in increasing the mechanical strength, cellular proliferation and dissolution/resorption rates [43, 44]. Moreover, the presence of magnesium in these materials favours bone growth, promoting osteogenic differentiation of preosteoblasts and improving osteointegration during the early stages of bone healing. During the
With the aim to convert waste into value-added products agricultural wastes (such as beer bagasse) have been investigated as potential renewable raw materials to develop bone scaffolds capable to support osteoblast growth for bone regeneration applications. Materials prepared here with residues from beer production contain P, Si, Ca and Mg as main components, which are also cations present in bone [11, 48]. Furthermore, the use of agricultural wastes to provide renewable raw materials for more advanced applications is of great interest giving value-added products that may lead to a significant reduction in waste accumulation. Moreover, due to their origin these materials are very competitive in price . The materials derived from beer bagasse (BBM) were biphasic calcium-magnesium phosphates with silica that can be either amorphous or as cristobalite, its crystallinity increasing in accordance with the final heat treatment temperature employed, with porosities that lie within the 10 to 100 µm range. All of these characteristics were important for the promotion of both cell proliferation and differentiation .
Osteogenic cells MC3T3-E1 are widely employed to study
The present work employs residues from beer production from three different Spanish plants, Lerida, Guadalajara and Burgos from the Mahou San Miguel group. These residues were chosen so that a comparison of their suitability and any effects of the differences in their chemical compositions on their cytocompatibility for bone growth could be determined. The beer bagasses were first dried at 150°C for 4 h, at a heating rate of 5 °C/min, in order to avoid putrefaction, due to their high humidity (70–85 wt%). Thermal stabilities of the dried materials were determined by TG-DTA analyses in air, to assess the temperature necessary to eliminate the organic matter and prepare stable and reproducible materials. Results from TG-DTA indicated that the thermal behaviour of the three samples under air treatment was practically identical, with total weight losses found of
The samples of bagasse were then calcined at 600, 700, 850 or 1000 °C, maintaining the final temperature for 4 h, the samples thus produced were designated as BBM46, BBM47, BBM48 and BBM410, respectively. The calcined materials were homogenised and their particle sizes controlled by being milled to less than 120 µm, due to the importance of this parameter in the reproducibility of biological behaviour of the materials.
The composition of the calcined materials was analysed by means of inductively coupled plasma atomic emission spectroscopy (ICP), showing four main elements that depending on the source of the beer bagasse were 15–20 % silicon, 12–14 % phosphorous, 7–8 % calcium and 5–7 % magnesium. The variations between different batches of beer bagasse from the same source were negligible and within experimental error. X-ray diffraction (XRD) patterns of samples showed that with higher heat-treatment temperatures the XRD peaks were narrower and better defined due to the increased crystallinity of the materials. The most significant crystalline phases were calcium-magnesium phosphate (*) (31.5 ° (100 %), 29.7 ° (85 %) and 29.3 ° (75 %) present at all temperatures and cristobalite which was only found when heat treatment temperatures greater than 600 °C were employed (21.9 ° (100 %, (111)), 36 ° (12 %, (220)), 31.3 ° (10 %, (102)) and 28.4 ° (8 %, (111)) [50, 51].
No crystalline cristobalite was found for BBM46 but mean crystallite sizes of 56 to 70 nm, 60 to 85 nm and 85 to 230 nm were found for BBM47, BBM48 and BBM410, respectively (Figure 16).
The three beer bagasses as received had identical FTIR traces and the heat treated materials prepared from them also displayed identical results. For the bagasse dried at 150 °C the principal bands were due to a broad band of O-H stretching in the 3100-3600 cm-1 region, the C-H aliphatic axial deformation in CH2 and CH3 groups from cellulose, hemicellulose and lignin at 2926 cm-1, the -OCH3 vibration at 2854 cm-1 due to lignin or hemicelluloses. The C=O stretching of the acetyl groups present in cellulosic material at 1743 cm-1, while the bands at 1043 and 1160, corresponded to O-H stretching of primary and secondary alcohols, respectively and the band at 1378 cm-1 corresponded to O-H vibration of phenolic groups. The signal at 899 cm-1 was assigned to β-glycosidic linkages between monosaccharide units. For the heat treated materials the broad band at 3466 cm-1 was due to the stretching vibration of the P-OH and Si-OH groups that was diminished with respect to the dried materials due to the loss of these groups on heating. The bands at 1164, 1121 and 1097 cm-1 were due to the Si-O-Si asymmetric stretching vibration, a band at 470 cm-1 associated with a network O-Si-O bond bending modes. The band at 1023 cm-1 was attributed to the symmetric terminal P-O stretching mode of the calcium magnesium phosphate, a band for asymmetric bridge P-O stretching mode appeared at 964 cm-1 whilst those at 579 and 496 were due to the asymmetric bending vibrations of terminal P-O bands [52-54].
The porosities and particle size distributions of the heat treated materials were determined by Mercury Intrusion Porosimetry. From Figure 17 it may be appreciated that the majority of the intrusion curve was due to interparticulate pore filling and that only with the materials treated at the lowest temperature was there any sign of mesoporosity, pores with diameters lower than 50 nm, due to intraparticulate porosity, which due to sintering of the materials disappeared on heating at higher temperatures.
As the heat treatment temperature was raised the density of the materials increased with a corresponding reduction in the cumulative pore volume accompanied by a slight displacement of the curves to wider pores. For finely divided powder samples the cumulative intrusion curve represents the void filling between the aggregates of the primary particles. Thus, an evaluation of their size may be made using the Mayer Stowe theory that relates the porosity of the sample to a packing factor, assuming spherical particle geometry, which is used to estimate the particle size from the measured width of the spaces between the particles. It may be observed from the results that higher heat treatment temperatures caused an increase in the aggregate sizes and a densification of the materials due to sintering of the samples, which was in agreement with the results observed from the XRD analyses of these materials [55,56].
Analyses of the basic character of materials by decomposition of acetic acid (Figure 18) indicate higher amount of basic sites for those prepared at lower temperatures, agreeing with the sintering process observed by the other characterisation techniques.
The osteoblast-like MC3T3-E1 murine cells were cultured in α-MEM (Gibco) that was supplemented with 10 % foetal bovine serum and 1 % penicillin-streptomycin (basal medium). In order to induce differentiation the cells were placed in osteogenic media: a basal medium supplemented with 10 mM β-glycerophosphate and 50 μg/mL ascorbic acid. These cells were incubated at 37 °C in a humidified atmosphere and at 5 % CO2. The cell culture results obtained were compared with a reference material, hydroxyapatite (HA), a synthetic calcium phosphate ceramic that mimics the natural apatite composition of bones and teeth and has been described as a potential material to coat scaffolds for promoting osteoblast differentiation [55,56].
Cell proliferation assays were performed in the presence of increasing concentrations of BBM derived materials from Lerida, Guadalajara and Burgos treated at increasing temperatures, after culturing cells in basal medium for 7 days, in order to determine the influence of the origin of the bagasses and the effect of the temperature to which they were subjected. To evaluate the proliferation rate of MC3T3-E1 cells grown in the presence of BBM derived materials, the cell viability was measured following incubation of the cells with materials at various concentrations, ranging from 20-200 µg/mL for 7 days. HA was used at the same concentrations as a reference material. To carry out the viability assays the cells were seeded into 96-well plates (10000 cells per well; four replicates for each condition). After 24 h, the cells were treated with materials for the specified concentrations and time periods. The cultures were then washed twice in phosphate buffered saline to remove any residual material. Subsequently, the tetrazolium dye, 3-(4,5-dimethilthiazol-2)-2,5-diphenyl-2H tetrazolium bromide (MTT, 5 mg/mL in phosphate-buffered saline; Sigma), was added to the medium and left for 1 h. Following removal of the medium, the precipitated formazan crystals were dissolved in optical grade dimethyl sulphoxide (200 μL). Then by use of an ELX808 microplate reader (BioTeK) the absorbance of each well was measured spectrophotometrically at 570 nm. When beer bagasses were treated at lower temperatures a decrease in cell proliferation rates was observed (Figure 19).
We found that the addition of BBM derived materials to MC3T3-E1 cells did not considerably alter the viability of the cells, compared to the reference material (HA). When BBM derived materials were subjected to 1000 °C for 4 h, proliferation analysis assessed by MTT test showed a similar cell growth to that obtained when using HA as reference material. However, a decrease in cell proliferation rates was observed when BBM derived materials were treated at low temperatures (600 °C). The observed delays in cell proliferation were due to the more basic pH for the powder containing media at the first day in culture, which could initially result in a lower cellular enzymatic efficiency and hence in slower processes (e.g. cell division and metabolism) than that observed with the control cells, growing on polystyrene plates . The different relative metabolic levels found in MC3T3-E1 cells growing in the presence of BBM derived materials at day 7 also correlate with the characteristics of the powders, where materials pretreated at lower temperatures produced higher increases in the pH of the culture medium due to their greater basicities, leading to lower proliferation rates of the MC3T3-E1 cells.
According to these findings, further cytotoxicity assays and cell differentiation experiments were performed on the BBM derived biomaterials treated at 1000 °C.
The cytotoxicity of the culture media was related to the lactate dehydrogenase (LDH) activity. The measurements were determined on cells plated at a density of 10 000 cells per well in 96-well plates in basal and osteogenic medium. Beer bagasse materials treated at 1000 °C for 4 h were added at 100 µg/mL. After 24 h, the culture media were collected and centrifuged and the supernatant was used for the LDH activity assay. The LDH activity was determined spectrophotometrically using the Cytotoxicity Detection kit (Roche), according to the manufacturer's instructions. Cells cultured in the presence of BB-derived materials showed no obvious cytotoxicity compared to cells grown in the presence of hydroxyapatite and polystyrene culture plates, used as controls (Figure 20).
Results obtained in LDH assay when MC3T3-E1 cells were cultured in the presence of BBM derived materials for 24 h support the cytocompatibility of these new materials.
Previous studies have demonstrated that expression of osteoblastic markers in MC3T3-E1 cells begins after culturing the cells with medium supplemented with β-glycerol-phosphate and ascorbic acid . The effects of direct contact of BBM derived materials and osteoblast-like cells in terms of cell differentiation were evaluated by testing alkaline phosphatase (ALP) activity, collagen production and extracellular matrix mineralisation after 15 days in culture. Alkaline phosphatase activity (ALP) begins to be expressed after 1 week and reaches a maximum after 2 weeks when MC3T3-E1 cells are cultured in osteogenic medium .
The capacity of cells growing in the presence of BB-derived materials was evaluated to express alkaline phosphatase, an early marker of osteoblastic cell differentiation. To this end, MC3T3-E1 cells were seeded into 96-well plates (10 000 cells per well; four replicates for each condition) and grown on basal and osteogenic medium for 15 days in the presence of BB-derived materials at 100 µg/ml. After treatment with the BBM derived materials, the cells were rinsed with PBS and then lysed into PBS containing 0.1 % Triton X-100. These cell lysates were then centrifuged and the soluble fraction used for the enzyme assay. The samples were first incubated with an assay mixture of
It was found that the ALP activity was higher in cells grown in osteogenic medium than for cells cultured in basal medium, as we would predict (Figure 21). However, there was no significant difference in the ALP activity observed for MC3T3-E1 cells grown in the presence of HA and with control cells. These results established that the presence of BB-derived materials did not affect ALP activity.
Results indicated that MC3T3-E1 cells maintain their capability to express active ALP enzymes when growing in the presence of BBM derived powders.
The addition of ascorbic acid in MC3T3-E1 cells is known to induce the deposition of collagen in the extracellular matrix . To confirm that osteoblastic cells exposed to the BB-derived materials indeed maintained the ability to differentiate at similar levels to control cells, the profile of type-I collagen cellular secretion, the main extracellular matrix protein expressed in bone, was also analysed. Collagen secretion by MC3T3-E1 cells cultured in the presence of BB-derived materials treated at 1000 °C for 4 h, at 100 µg/mL was quantified by Sirius Red staining. After culturing MC3T3-E1 cells in the presence of BB-derived materials for 15 days in both basal and osteogenic media, the cells were washed three times with PBS and then fixed in 4 % paraformaldehyde. Following the three rinses in PBS, the cell cultures were stained for collagen secretion in a 0.1 % solution of Sirius Red (Sigma) in saturated picric acid for 18 h. Following washing with 0.1 M acetic acid until the disappearance of the red colour, the stain on specimens was eluted in destain solution (0.2 M NaOH–methanol 1:1). The optical density at 540 nm was then determined using a spectrophotometer.
From the results shown in Figure 22, it may be seen that collagen deposition was promoted when MC3T3-E1 cells were grown in osteogenic medium at all the tested conditions, as expected and no significant differences in the collagen production were observed for cells grown in the presence of BB-derived materials compared with those grown on plastic plates, neither in basal nor osteogenic medium.
Extracellular matrix mineralisation is also one of the major aspects of bone formation. Minerals formed
Extracellular matrix calcium deposits for mineralised nodule formation were stained with Alizarin red S dye which combines with calcium ions. After culturing MC3T3-E1 cells in the presence of BBM derived materials at 100 µg/mL for 15 days, the cells were then washed thrice with PBS and subsequently fixed in 75 % ethanol for 1 h. These cell cultures were then stained with 40 mM Alizarin Red S in distilled water (pH 4.2) for 10 min at room temperature. The cell monolayers were then washed with distilled water until no more colour appeared. The stain was dissolved in 10 % cetylpyridinium chloride in 10 mM sodium phosphate (pH 7.0) and the absorbance values at 620 nm were measured. The extracellular matrix mineralisation determined by Alizarin Red S staining is shown in Figure 23. In all of the tested conditions cells grown in osteogenic medium for 15 days displayed slightly higher calcium content, an indicator of mineralisation nodule formation. Treatment with BB-derived materials at a concentration of 100 μg/mL for 15 days did not significantly affect the mineralisation rates compared with those of control cells grown on polystyrene plates (Figure 23a). For these determinations HA (a material which contains Ca/P-apatite) could not be used as a control due to the high background produced by the material itself. These results indicated normal mineralisation induced by MC3T3-E1 cells in long-term cultures was not affected by the BB-derived materials.
Overall, in the presence of BB derived materials the osteoblast functions displayed normal cell differentiation profiles with respect to alkaline phosphatase activity, collagen secretion and extracellular matrix mineralisation. Furthermore, these parameters were maintained when compared to control cells grown on plastic plates and also with those obtained by culturing MC3T3-E1 cells in the presence of the same amounts of HA. The use of these BB-derived materials as coatings of metallic and ceramic bioimplants for odontoestomatologic treatments, or to form part of 3D scaffolds with pore sizes designed with the desired characteristics for bone replacement, is currently under study. Together the results suggest that the developed materials may potentially be used to prepare scaffolds for bone tissue engineering applications.
5.2. Controlled desorption of bioactive substances
The biological activity of a substance depends mainly on the nature of its interaction with the tissue or organ; it must reach the target in an amount that is adequate to produce the desired effect, which means that it should be liberated in the particular place at a controlled rate. The same amount of active ingredient can have different effects when it is formulated as oral solution or in capsules or pills, due to the different rates of adsorption of the active agents in the digestive track. These requirements mean that preparing drugs from pure substances is a multidisciplinary and extensive field that requires multidisciplinary expertise in pharmaceutical sciences, engineering, material sciences, physical chemistry, polymer science, solution chemistry and biochemistry, amongst others.
The work presented here is based on the use of beer and rice production residues to prepare materials with special characteristics towards controlled desorption of bioactive substances, using the anticarnogenic drug 5-Fluorouracil as the model molecule (Figure 24).
The drug 5-fluorouracil (5-FU) is a pyrimidine analogue, used in the treatment of cancer, as it is an irreversible inhibitor of thymidylate synthase; interrupting the action of this enzyme blocks synthesis of the pyrimidine thymidine, nucleoside required for DNA replication . 5-FU belongs to the World Health Organization's List of Essential Medicines, being part of the family of drugs called antimetabolites. It has been used amongst others in the treatment of breast, stomach, pancreatic and skin cancers. Its main disadvantage is that the same dose of 5-FU may have therapeutic response with low toxicity in some patients, while even life-threatening toxicity in others . Thus, its use in a controlled manner is of great interest to avoid these problems. Parenteral administration causes a rapid elimination of 5-FU with an apparent terminal half-life of approximately 8-20 min. Choosing a proper controlled release system can improve its anticancer activity and also decrease the adverse side effects. 5-FU is a neutral weak acid [62, 63] whose tautomers structures are shown in Figure 25.
The design of materials to be used in desorption of bioactive substances is based on a thorough study of the conditions necessary to achieve a texture and structure capable to induce desorption in a controlled manner. The residues were first treated thermally to avoid putrefaction, as indicated above and then, according to TG-DTA analyses three different temperatures were chosen,
The biocompatibility of these bioecomaterials BBM47, BBM48 or BBM410 was studied after crushing and sieving, and homogeneized to a controlled particle size to favour reproducible results, according to previous studies. In order to assess the bioecomaterials biocompatibility a human glioblastoma cell line (1321N1) was used (Figure 26).
The viability of 1321N1 cells growing in the presence of the materials was determined at 1, 3 and 7 days.The quantification of cell viability on the different materials (Figure 27) indicates that all the materials were biocompatible, with cell viabilities similar or even better than the plastic control up to 7 days.
The preparation of the bioecomaterials to be used in controlled desorption was undertaken by studying different important parameters in their structuration, such as pelletising conditions, use of porogens,
After the pellets were prepared, they were sintered, once again after the corresponding study of the temperature and time of sintering, since they are parameters of utmost importance. Experimental results indicated that the optimum temperature of sintering was 700 °C for 4 h. Lower temperatures or times did not produce pellets with enough cohesion and higher temperautres or times produced materials with lower surface areas and pore volumes.
To determine the 5-FU concentration needed to eliminate 1321N1 cells after 2 days in cultures, the MTT assay was used, as described above. The 1321N1 cells were cultured (20000 cells/well in 24-well plates) at 37 °C in 5 % CO2 with 95 % humidity atmosphere for 24 h to favour their adhesion to the plates. Afterwards different concentrations of 5-FU were added to the medium and 2 days later the MTT assay was carried out.
From the results shown in Figure 28, it can be observed that a 5-FU concentration of 10 μg/mL was enough to eliminate all 1321N1 cells present in the culture. However, a concentration of 20 μg/mL was chosen, since it was possible that not all the adsorbed 5-FU would be liberated. Consequently, 200 μL of a solution 5-FU (20μg/mL) were added to each pellet, left to dry at room temperature for 24 h, protected from light, 5-FU is light sensitive. Finally the pellets were left in contact with human glioblastoma cells 1321N1, growing in DMEM (Gibco) supplemented with 10 % of bovine phoetal serum and 1 % peniciline/estreptomicine and the cell viability studied with fluorescent probes, as indicated above, following the decrease in cell viability as the 5-FU was desorbed from the pellets. The analyses of the desorption kinetics was carried out by comparison with the data obtained using a mesoporous silica prepared in our research group from rice husk (MR). This mesoporous material contains more than 97% silica, which is considered as ideal for desorption of pharmaceutical compounds .
According to these results, it can be said that 1321N1 cells could be eliminated with both materials, although BBM47 eliminates cancerous cells in a more controlled manner than MR. The characterisation of these materials indicates that MR was an amorphous mesoporous silica with a surface area of 98 m2g-1 and BBM was crystalline with silica in the form of cristobalite and calcium and magnesium phosphate of 4 m2g-1, also the basicity of BBM was much higher than that of MR, given its composition rich in alkaline earth cations (Figures 13 and 18), versus MR having more than 97% silica. The importance of the basic properties of the materials for the controlled release of 5-FU was to be expected, since given the acidity of 5-FU it would adsorb on basic sites. This explains why even though BBM47 had a much lower surface area than MR, its much higher basicity leads to a greater interaction with 5-FU, facilitating its controlled desorption, after careful design of its textural and structural characteristics [62, 65-67].
Residues from the beer, rice and sunflower oil production industries were used to prepare materials capable of cleaning wastewaters, support enzymes, act as catalysts or as scaffolds for tissue engineering or controlled desorption of bioactive substances. The use of these waste materials gives them an added value as a sustainable, environmentally friendly and economic supply of nanostructured materials,
The authors wish to acknowledge the financial support from the CDTI projects “Systems to recycle aqueous effluents with valorisation of contaminants and reduction of discharges. Total oxidation of contaminants in effluents by using Ecomaterials” (CDTI IDI-20091139), “Development of applications of glycerine sub product of biodiesel with catalysts made from Ecomaterials” (CDTI IDI-20121298) and “Design of sílica with controlled texture to be used in detergency using as raw material rice husk ash” (CDTI IDI-20143711-20143713) and the Spanish National Science Council for the projects “Valorisation of agriresidues for generation of biocompatible materials (Ecomaterials) to be used in dental implants” (INNPACT IPT-2011-1935-310000), “Preparation of materials from agro industrial residues for biomedical uses” (PIE CSIC 201480E103) and “Valorisation of Spanish agriresidues for advanced uses” (PIE CSIC 201460E105).
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