**5. Bioecomaterials**

#### **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 [39]. 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 mesen‐ chymal stem cells to differentiate into preosteoblasts to begin the bone-forming process [40].

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 [41]. 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 [42]. Calcium phosphate-based scaffolds exhibit osteoconductivity, bioactivity and resorbability *in vivo* due to their complex chemical composition (Ca/P ratio) and physical properties such as crystallo‐ graphic structure and porosity [40]. However, major drawbacks in the use of synthetic calcium phosphates are their price and use of non-renewable resources.

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 *in vivo* degradation of the scaffolds cell proliferation and differentiation are promoted by the release of their component elements [45-47].

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

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 [39]. 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

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 mesen‐ chymal stem cells to differentiate into preosteoblasts to begin the bone-forming process [40].

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 [41]. 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 [42]. Calcium phosphate-based scaffolds exhibit osteoconductivity, bioactivity and resorbability *in vivo* due to their complex chemical composition (Ca/P ratio) and physical properties such as crystallo‐ graphic structure and porosity [40]. However, major drawbacks in the use of synthetic calcium

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

and environmental impact of these processes [37, 38].

**5.1. Tissue engineering (Dental and Bone replacement therapies)**

phosphates are their price and use of non-renewable resources.

**5. Bioecomaterials**

202 Agroecology

implanted devices.

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 compo‐ nents, 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 [49]. 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 [50].

Osteogenic cells MC3T3-E1 are widely employed to study *in vitro* matrix mineralisation, since these cells can differentiate into osteoblasts that express strong ALP activity and can form a collagenous matrix organised in 3-dimensional nodules, which in the presence of ascorbic acid and phosphate progressively become mineralised [51]. The MC3T3-E1 cells display a timedependent sequential expression of osteoblast characteristics that are analogous to *in vivo* bone formation [52]. It has been shown that some biomaterials are able to modify directly the osteoblastic proliferation rate and its differentiation, such as the synthesis of alkaline phos‐ phatase, matrix mineralisation and collagen secretion [53,54]. Thus, the *in vitro* proliferation and differentiation responses of this osteoblast like cell line (MC3T3-E1) to the BBM derived powders were studied. Several biological responses to the biomaterials were assayed, includ‐ ing determinations of cell viability by the MTT and LDH assays, evaluation of ALP activity, Type- I collagen secretion and evaluation of matrix mineralisation at the differentiation period.

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 *ca*. 97 % of the initial mass, 8 % due to loss of volatile matter and water at T < 200 °C, 55 % loss at T = 200-380 °C caused by the decomposition of organic matter (mainly cellulose and hemicellulose) and finally a 34 % lost for T = 380-600 °C, corresponding to the decomposition of lignin [48,49]. For a more detailed explanation the results obtained with sample BBM Lerida are shown in Figure 15b.

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 *in vivo* degradation of the scaffolds cell proliferation and differentiation are promoted by the release of their component elements [45-47].

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 [49]. 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

Osteogenic cells MC3T3-E1 are widely employed to study *in vitro* matrix mineralisation, since these cells can differentiate into osteoblasts that express strong ALP activity and can form a collagenous matrix organised in 3-dimensional nodules, which in the presence of ascorbic acid and phosphate progressively become mineralised [51]. The MC3T3-E1 cells display a time-dependent sequential expression of osteoblast characteristics that are analogous to *in vivo* bone formation [52]. It has been shown that some biomaterials are able to modify directly the osteoblastic proliferation rate and its differentiation, such as the synthesis of alkaline phosphatase, matrix mineralisation and collagen secretion [53,54]. Thus, the *in vitro* proliferation and differentiation responses of this osteoblast like cell line (MC3T3-E1) to the BBM derived powders were studied. Several biological responses to the biomaterials were assayed, including determinations of cell viability by the MTT and LDH assays, evaluation of ALP activity, Type- I collagen

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 150ºC for 4 h, at a heating rate of 5 ºC/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

samples under air treatment was practically identical, with total weight losses found of *ca*. 97 % of the initial mass, 8 % due to loss of volatile matter and water at T < 200 °C, 55 % loss at T = 200-380 °C caused by the decomposition of organic matter (mainly cellulose and hemicellulose) and finally a 34 % lost for T = 380-600 °C, corresponding to the decomposition of lignin [48,49]. For a

characteristics were important for the promotion of both cell proliferation and differentiation [50].

more detailed explanation the results obtained with sample BBM Lerida are shown in Figure 15b.

secretion and evaluation of matrix mineralisation at the differentiation period.

**Figure 15**. Reproducibility of beer bagasse from a) Lerida, Guadalajara and Burgos and detailed analysis of b) BBM-Lerida. The samples of bagasse were then calcined at 600, 700, 850 or 1000 ºC, 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 **Figure 15.** Reproducibility of beer bagasse from a) Lerida, Guadalajara and Burgos and detailed analysis of b) BBM-Lerida.

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 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.

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]. 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, respec‐

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 *in vivo* degradation of the scaffolds cell proliferation and differentiation are promoted by the release of their component elements [45-47].

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 [49]. 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

Osteogenic cells MC3T3-E1 are widely employed to study *in vitro* matrix mineralisation, since these cells can differentiate into osteoblasts that express strong ALP activity and can form a collagenous matrix organised in 3-dimensional nodules, which in the presence of ascorbic acid and phosphate progressively become mineralised [51]. The MC3T3-E1 cells display a time-dependent sequential expression of osteoblast characteristics that are analogous to *in vivo* bone formation [52]. It has been shown that some biomaterials are able to modify directly the osteoblastic proliferation rate and its differentiation, such as the synthesis of alkaline phosphatase, matrix mineralisation and collagen secretion [53,54]. Thus, the *in vitro* proliferation and differentiation responses of this osteoblast like cell line (MC3T3-E1) to the BBM derived powders were studied. Several biological responses to the biomaterials were assayed, including determinations of cell viability by the MTT and LDH assays, evaluation of ALP activity, Type- I collagen

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 150ºC for 4 h, at a heating rate of 5 ºC/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 *ca*. 97 % of the initial mass, 8 % due to loss of volatile matter and water at T < 200 °C, 55 % loss at T = 200-380 °C caused by the decomposition of organic matter (mainly cellulose and hemicellulose) and finally a 34 % lost for T = 380-600 °C, corresponding to the decomposition of lignin [48,49]. For a

characteristics were important for the promotion of both cell proliferation and differentiation [50].

more detailed explanation the results obtained with sample BBM Lerida are shown in Figure 15b.

a) b)

(100 %, (111)), 36 ° (12 %, (220)), 31.3 ° (10 %, (102)) and 28.4 ° (8 %, (111)) [50, 51].

reproducibility of biological behaviour of the materials.

(220)), 31.3 ° (10 %, (102)) and 28.4 ° (8 %, (111)) [50, 51].

biological behaviour of the materials.

Lerida.

204 Agroecology

**Figure 15**. Reproducibility of beer bagasse from a) Lerida, Guadalajara and Burgos and detailed analysis of b) BBM-Lerida.

**Figure 15.** Reproducibility of beer bagasse from a) Lerida, Guadalajara and Burgos and detailed analysis of b) BBM-

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

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 %,

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, respec‐

The samples of bagasse were then calcined at 600, 700, 850 or 1000 ºC, 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

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 °

secretion and evaluation of matrix mineralisation at the differentiation period.

**Figure 16.** XRD patterns of Lerida agriresidue derived materials heat treated at 600 °C a, 700 °C b, 850 °C c and 1000 °C d.

tively 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 disap‐ peared 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].

**Figure 17.** Mercury intrusion porosimetry results for beer bagasse from Burgos treated for 4 h at 700 °C, 850 °C or 1000 °C.

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.

**Figure 18.** TGMS analyses of decomposition of acetic acid on BBM47 (grey line) and BBM410 (black line).

#### **Cell proliferation and differentiation on BBM derived materials**

#### **Cell cultures**

The osteoblast-like MC3T3-E1 murine cells were cultured in α-MEM (Gibco) that was supple‐ mented 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 on BBM derived materials**

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

**Figure 17.** Mercury intrusion porosimetry results for beer bagasse from Burgos treated for 4 h at 700 °C, 850 °C or 1000 °C.

**Figure 18.** TGMS analyses of decomposition of acetic acid on BBM47 (grey line) and BBM410 (black line).

The osteoblast-like MC3T3-E1 murine cells were cultured in α-MEM (Gibco) that was supple‐ mented 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

**Cell proliferation and differentiation on BBM derived materials**

**Cell cultures**

206 Agroecology

sintering process observed by the other characterisation techniques.

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).

**Figure 19.** MC3T3-E1 proliferation analysis by MTT assay on cells treated with different concentrations of BBM de‐ rived materials treated at several temperatures after one week in culture. The data were analysed by single factor anal‐ ysis of variance followed by the *post hoc* Tukey's honestly significant difference test, \*p<0.05 with respect to control (cells growing on polystyrene plates).

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 [57]. 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.

### **Cytotoxicity**

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 manu‐ facturer'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 polystyr‐ ene culture plates, used as controls (Figure 20).

**Figure 20.** Cytotoxicity study by lactate dehydrogenase (LDH) activity assay on MC3T3-E1 cells treated with 100 μg/ml of BBM derived materials treated at 1000 °C and HA, after 24 h in culture.

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.

#### **Cell differentiation**

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 [57]. 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

According to these findings, further cytotoxicity assays and cell differentiation experiments

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 manu‐ facturer'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 polystyr‐

Control Lérida Guadalajara Burgos HA

derived materials for 24 h support the cytocompatibility of these new materials.

**Figure 20.** Cytotoxicity study by lactate dehydrogenase (LDH) activity assay on MC3T3-E1 cells treated with 100 μg/ml

Results obtained in LDH assay when MC3T3-E1 cells were cultured in the presence of BBM

BASAL OSTEOGENIC

were performed on the BBM derived biomaterials treated at 1000 °C.

ene culture plates, used as controls (Figure 20).

0,00

of BBM derived materials treated at 1000 °C and HA, after 24 h in culture.

0,10

0,20

Absorbance

490nm (a.u.)

0,30

0,40

0,50

cells.

208 Agroecology

**Cytotoxicity**

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 [52]. 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 [52].

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 *p*-nitrophenyl phosphate (p-NPP) (Sigma). Cleavage of the *p*-NPP in a soluble yellow end product, *p*-nitrophenol, which absorbs at 405 nm, was used to assess the ALP activity. The optical density of *p*-nitrophenol at 405 nm was then determined spectrophoto‐ metrically and the ALP activities normalised to total protein content using the bicin-choninic acid (BCA) method. The ALP activity of each condition was quantified and compared to that present in cells grown on polystyrene plates used as the (control).

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.

**Figure 21.** MC3T3-E1 differentiation study by alkaline phosphatase (ALP) activity assay on cells treated with 100 μg/ml of BB-derived materials and HA after 2 weeks in culture.

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 [52]. 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 BBderived 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.

**Figure 22.** MC3T3-E1 differentiation study by collagen production (Sirius Red staining) on cells treated with 100 μg/ml of BB-derived materials and HA after 2 weeks culture.

Extracellular matrix mineralisation is also one of the major aspects of bone formation. Minerals formed *in vitro* were found to consist of calcium and phosphorus deposited on well-bonded collagen fibrils, and some of the crystals matured into hydroxyapatite crystals [58]. Besides its effect on ALP activity and collagen synthesis, we determined whether BB-derived materials might affect the mineralisation of the matrix formed by MC3T3-E1 cells. These cells are known to deposit minerals in the collagenous matrix in the presence of β-glycerol phosphate [59].

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 deter‐ mined 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 BBderived materials. 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 AR). 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.

scaffolds for bone tissue engineering applications.

**5.2 Controlled desorption of bioactive substances**

Results indicated that MC3T3-E1 cells maintain their capability to express active ALP enzymes

The addition of ascorbic acid in MC3T3-E1 cells is known to induce the deposition of collagen in the extracellular matrix [52]. 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 BBderived 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

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,

**Figure 22.** MC3T3-E1 differentiation study by collagen production (Sirius Red staining) on cells treated with 100 μg/ml

Extracellular matrix mineralisation is also one of the major aspects of bone formation. Minerals formed *in vitro* were found to consist of calcium and phosphorus deposited on well-bonded collagen fibrils, and some of the crystals matured into hydroxyapatite crystals [58]. Besides its effect on ALP activity and collagen synthesis, we determined whether BB-derived materials might affect the mineralisation of the matrix formed by MC3T3-E1 cells. These cells are known to deposit minerals in the collagenous matrix in the presence of β-glycerol phosphate [59].

when growing in the presence of BBM derived powders.

210 Agroecology

then determined using a spectrophotometer.

neither in basal nor osteogenic medium.

of BB-derived materials and HA after 2 weeks culture.

**Figure 23.** a) MC3T3-E1 differentiation study by extracellular matrix mineralisation (Alizarin Red S staining) on cells treated with 100 µg/ml of BBM derived materials after 2 weeks culture.b) Osteoblasts MC3T3-E1growing on BBM46, nucleus tinted in blue (Hoechst) and actin microphilaments of citoeschelet in red (Phalloidine) Overall, in the presence of BB derived materials the osteoblast functions displayed normal cell differentiation profiles with respect to **Figure 23.** a) MC3T3-E1 differentiation study by extracellular matrix mineralisation (Alizarin Red S staining) on cells treated with 100 μg/ml of BBM derived materials after 2 weeks in culture. b) Osteoblasts (MC3T3-E1cells) growing in the presence of BBM derived materials. Cells were stained with phalloidine (red) and the nuclei were counterstained with ToPro-3 (blue).

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

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 medicines 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 anticarnogen 5-Fluorouracil as the model molecule (Figure 24).

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 pharmaceut‐ ical 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).

**Figure 24.** 5-Fluorouracil, anticarcinogen

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 [60]. 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 lifethreatening toxicity in others [61]. 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.

**Figure 25.** 5-FU tautomers

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.

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 pharmaceut‐ ical sciences, engineering, material sciences, physical chemistry, polymer science, solution

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,

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 [60]. 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 lifethreatening toxicity in others [61]. 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

using the anticarnogenic drug 5-Fluorouracil as the model molecule (Figure 24).

**5.2. Controlled desorption of bioactive substances**

212 Agroecology

chemistry and biochemistry, amongst others.

**Figure 24.** 5-Fluorouracil, anticarcinogen

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 desorp‐ tion 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, *i.e.* 700, 850 and 1000°C, after previous studies by infrared spectroscopy (FTIR), textural analysis, X-ray diffraction and acetic acid decomposition on basic sites by TG-MS, and textural analyses by N2 adsorption/desorption and mercury intrusion porosimetry. In this way the residue derived materials have different structure and surface characteristics, with higher crystallinities and lower surface areas, porosities and basic sites on their surfaces, on increasing the treatment temperature.

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).

**Figure 26.** Images of 1321N1 cells stained with AM-calcein showing live cells.

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.

**Figure 27.** Cell viability (%) determined by MTT assay showing cells growing on biomaterials after 1, 3 and 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, *etc.*. The particle size was controlled by sieving, and the conditions of pelletising (1 cm diameter) studied by changing the amount of material, pressure and time, being optimised at 5 tons pressure for 2 min. The material used was BBM47, since this had a higher surface area and porosity leading to a greater capacity for 5-FU absorption.

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) supple‐

Effluent Cleaning, Greener Catalysts and Bioecomaterials from Agricultural Wastes http://dx.doi.org/10.5772/60018 215

**Figure 28.** Cell viability after 2 days of treatment with different amounts of 5-FU (μg/mL).

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

**Figure 27.** Cell viability (%) determined by MTT assay showing cells growing on biomaterials after 1, 3 and 7 days.

higher surface area and porosity leading to a greater capacity for 5-FU absorption.

and 2 days later the MTT assay was carried out.

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, *etc.*. The particle size was controlled by sieving, and the conditions of pelletising (1 cm diameter) studied by changing the amount of material, pressure and time, being optimised at 5 tons pressure for 2 min. The material used was BBM47, since this had a

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

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) supple‐

plastic control up to 7 days.

214 Agroecology

mented 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 [64].

**Figure 29.** Viability of 1321N1 cells after 1, 2, 3 or 7 days in contact with BBM47 bioecomaterial or MR with 5-FU adsorbed.

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 mesopo‐ rous silica with a surface area of 98 m2 g-1 and BBM was crystalline with silica in the form of cristobalite and calcium and magnesium phosphate of 4 m2 g-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].
