**4. Collagen/ modified layered silicate nanocomposites**

134 Materials Science and Technology

45 °C. Purified bentonite results (PB), which is ground to obtain a powder with < 60 µm size

Bentonite morphology, before and after purification, is highlighted through X-ray diffraction (XRD), thermo-gravimetrical analyses (TGA) and scanning electron microscopy

From X-ray diffractions we notice that small angle diffraction peaks present a higher surface area in purified bentonite as compared with the initial one, proving an increase of MMT concentration. We accepte that MMT and cristobalite are the two main components in the bentonite analysed (without taking into account the impurity components). We calculate the concentration of MMT in initial bentonite based on the maximum diffraction intensities: 77% montmorillonite in initial bentonite and 82% montmorillonite in purified bentonite (Vuluga

The TGA results (Table 2) are in accordance with X-ray diffraction results. We notice that, after purification the mass loss on the second step of decomposition decreases, while the mass loss on the third step of decomposition increases proving the decrease of the

> II 200÷400°C

(PB) 8.3 - - 1.4

From scanning electron microscopy images (Fig. 3 and 4) we notice the morphology of purified powder with submicron dimensions and with uniform arrangement of chemical elements as compared to the morphology of initial powder where chemical elements are

Fig. 3. Secondary electron images (SEI) and distribution of X-rays for initial bentonite.

Initial bentonite (B) 6.8 0.6 0.4 1

Weight loss on each decomposition step (%)

III 400÷550°C

IV 550÷700°C

impurities concentration and the increase of the amount of montmorillonite.

I 20÷200°C

Table 2. TGA results for bentonite before and after purification.

particles.

(SEM).

et al., 2008b).

Sample

Purified bentonite

relatively uniformly dispersed.

Polymer/inorganic nanocomposites have attracted great recent attention and possessed a special position in constructing nano-assemblies due to their unique microstructure, outstanding properties and particular versatility. Nanocomposite materials can achieve much better properties than just the sum of their components as a result of interfacial interaction between the matrix and filler particles. It is the nature and degree of such interactions that play a pivotal role on the characteristics of resulted nanocomposites such as solubility, optical properties, electrical and mechanical aspects, biocompatibility, biodegradability. The organic-inorganic composites/hybrids represent a recent topic in which bibliographic references are still few. The year 2000 could be considered as the "starting point" in this field. Research for obtaining new types of bio-nanocomposites based on collagen in solid physical forms of matrices or membranes in which inorganic components can be hydroxyapatite, biovitroceramica, TiO2, SiO2, natural layered silicate as it is or modified with hydrolysed collagen, antioxidants has developed (Ficai et al. 2010a; Albu et al., 2009; Baia et al., 2008; Piticescu et al., 2008; Potarniche et al., 2010; Vuluga et al., 2007; Vuluga et al., 2008a). Organic compounds that can interact with collagen are represented by a series of biological active substances or other biocompatible polymers, their compatibility being previously tested with collagen gel or colloidal solutions. In such phases, the nanocomposites are obtained: membranes by free drying at 25 °C or matrices by freeze-drying process ( lyophilization). According to the type of obtained nanocomposite, the amount of non collagen components should be: in case of matrices between 5÷20% insoluble powders and 30÷50% water soluble substances; in case of membranes between 1÷2% insoluble powders and 10-30% water soluble substances. Exceeding these amounts negatively influences the collagen matrix and membrane specific stability structure as well as the mechanical and hydrophilic properties. (Olteanu et al., 2008; Trandafir at al., 2007, Ficai et al., 2010b, Lungu et al., 2007, Titorencu et al., 2010).

Using hydrolyzed collagen modified layered silicates we obtain collagen based nanocomposites which can be used in healing varicose ulcer, the silicate improving the regenerative action of type I collagen on conjunctive tissue, reducing the healing time of ulcerous wound (Trandafir et al., 2007).

By introducing maleic copolymers as structural modifiers of layered silicates leads to binary stable hybrids compatible with collagen gel, a process which forms a liquid ternary nanocomposite resistant to further processing, for example to lyophilization.

Collagen - Modified Layered Silicate Biomaterials for Regenerative Medicine of Bone Tissue 137

From X-ray diffractions (Fig. 7 and 8) we notice that silicate structure changes in a specific way: a peak disappears at 2 θ = 23°, which is silicate characteristic peak and shapes very well the peak at approximately 2 θ = 3°. This proves the interaction between silicate and maleic copolymers as well as an organized structure. Intercalated nanocomposites are obtained with a lamellar ordered structure. Maleic copolymer with hydrophobic comonomer (MA-MMA) interacts better with silicate, the best structure is obtained at 1:1 silicate/copolymer ratio. In the case of maleic copolymer with hydrophilic comonomer (MA-VA), the best structure is obtained at a low amount of copolymer, meaning at 2:1 ratio. These two types of layered silicate/ maleic copolymer binary nanocomposites are selected

From thermal stability point of view, binary nanocomposites thermogrames present differences according to hydrophilic or hydrophobic comonomer used to modify layered

As compared to purified bentonite, at which the maximum rate of decomposition is on the first step, at a temperature of 74 °C, the organophilized bentonite presents a shift of maximum decomposition rate towards higher temperatures (220÷225°C for PB/ MA-VA nanocomposites and 220 ÷365 °C for PB/ MA-MMA nanocomposites), on the second step of decomposition, similar to maleic copolymers (Table 3). An increase of maleic copolymer concentration (from 50% to approximately 200% reported to bentonite) reflects into an increase of weight loss at the temperature range of 200 ÷500 °C. The obtained results show the intercalation of maleic copolymers between the silicate layers. Thermal stability of obtained binary nanocomposites correlates with the degree of interaction between silicate and copolymer. We notice that in the case of PB/ MA-MMA nanocomposites the best thermal stability is obtained at 1:1 ratio while the best thermal stability for the PB/ MA-VA nanocomposites is obtained at 2:1 ratio. These results are in accordance with X-ray

Fig. 6. Particle size and morphology for PB/ MA-VA binary nanocomposites.

to obtain ternary nanocomposites.

**Size of particles, (nm)**

silicate.

diffraction results.

**0 50 100 150 200 250 MA-VA copolymer, (%)**

### **4.1 Layered silicate/maleic copolymers binary nanocomposites**

Until now, there has not been shown any interest in obtaining systems/composites/hybrids based on copolymers of anhydride or maleic acid with clays/bentonites. Literature is mostly made of patents which refer to synthesis or uses of systems in which the bentonite and the polymer are mixed without being associated with the composites or hybrids formation. One of the first areas, in which they are mentioned as systems of bentonite and maleic copolymers or polymers in general, is conducting drilling fluids for oil secundary recovery (Al-Marhoun et al., 1988; Gleason & Brase, 1985; Hale & Lawson, 1988; Hayes, 2005; Jarm et al., 1988; Libor et al., 1987; Martinko, 1987; Shaarpour, 2004). In medical field, different systems of bentonite/maleic polymers are obtained for dentistry uses (Heathman & Ravi, 2005), for parasites control (mites, moths) and water or dirt rejection (De Sloovere et al., 2004) and as dirt retention agents recommended for hand cleaning pastes (Florescu et al., 1994). In conclusion, we can say that the composite/organo-inorganic hybrids based on bentonite and maleic copolymers are a promising open field.

The natural layered silicate/maleic copolymer binary nanocomposites are obtained by solution intercalation method (Ray & Okamoto, 2003). According to the chemical structure of maleic partner, the interaction between inorganic and organic components is performed at different ratios (1: 1; 1: 2 and 2: 1), in a mixture of 1:1 ethanol- water. Thus, we obtain maleic copolymer modified layered silicate binary nanocomposites: PB/ MA-MMA (1: 1; 1: 2 and 2: 1) and PB/ MA-VA (1: 1; 1: 2 and 2: 1).

From particle size analysis of resulted binary nanocomposites (Fig. 5 and 6) we notice:


Fig. 5. Particle size and morphology for PB/ MA-MMA binary nanocomposites.

Until now, there has not been shown any interest in obtaining systems/composites/hybrids based on copolymers of anhydride or maleic acid with clays/bentonites. Literature is mostly made of patents which refer to synthesis or uses of systems in which the bentonite and the polymer are mixed without being associated with the composites or hybrids formation. One of the first areas, in which they are mentioned as systems of bentonite and maleic copolymers or polymers in general, is conducting drilling fluids for oil secundary recovery (Al-Marhoun et al., 1988; Gleason & Brase, 1985; Hale & Lawson, 1988; Hayes, 2005; Jarm et al., 1988; Libor et al., 1987; Martinko, 1987; Shaarpour, 2004). In medical field, different systems of bentonite/maleic polymers are obtained for dentistry uses (Heathman & Ravi, 2005), for parasites control (mites, moths) and water or dirt rejection (De Sloovere et al., 2004) and as dirt retention agents recommended for hand cleaning pastes (Florescu et al., 1994). In conclusion, we can say that the composite/organo-inorganic hybrids based on

The natural layered silicate/maleic copolymer binary nanocomposites are obtained by solution intercalation method (Ray & Okamoto, 2003). According to the chemical structure of maleic partner, the interaction between inorganic and organic components is performed at different ratios (1: 1; 1: 2 and 2: 1), in a mixture of 1:1 ethanol- water. Thus, we obtain maleic copolymer modified layered silicate binary nanocomposites: PB/ MA-MMA (1: 1; 1:

From particle size analysis of resulted binary nanocomposites (Fig. 5 and 6) we notice:

the PB/ MA-MMA nanocomposite presents particle size dimensions at a range of

 the PB/ MA-VA nanocomposite presents a similar behavior as that obtained with PB/ MA-MMA the difference being the particle size is bigger (at a range of 4000÷13000 nm); the higher the maleic copolymer concentration is the bigger the particle size; this is valid up to an approximately 50% concentration as compared to silicate particle size; an over 50% concentration is followed by a decrease in particle size up to a close value of

Fig. 5. Particle size and morphology for PB/ MA-MMA binary nanocomposites.

**4.1 Layered silicate/maleic copolymers binary nanocomposites** 

bentonite and maleic copolymers are a promising open field.

2 and 2: 1) and PB/ MA-VA (1: 1; 1: 2 and 2: 1).

**0 50 100 150 200 250 MA-MMA copolymer, (%)**

3000÷6000 nm;

unmodified silicate.

**Size of particles, (nm)**

Fig. 6. Particle size and morphology for PB/ MA-VA binary nanocomposites.

From X-ray diffractions (Fig. 7 and 8) we notice that silicate structure changes in a specific way: a peak disappears at 2 θ = 23°, which is silicate characteristic peak and shapes very well the peak at approximately 2 θ = 3°. This proves the interaction between silicate and maleic copolymers as well as an organized structure. Intercalated nanocomposites are obtained with a lamellar ordered structure. Maleic copolymer with hydrophobic comonomer (MA-MMA) interacts better with silicate, the best structure is obtained at 1:1 silicate/copolymer ratio. In the case of maleic copolymer with hydrophilic comonomer (MA-VA), the best structure is obtained at a low amount of copolymer, meaning at 2:1 ratio. These two types of layered silicate/ maleic copolymer binary nanocomposites are selected to obtain ternary nanocomposites.

From thermal stability point of view, binary nanocomposites thermogrames present differences according to hydrophilic or hydrophobic comonomer used to modify layered silicate.

As compared to purified bentonite, at which the maximum rate of decomposition is on the first step, at a temperature of 74 °C, the organophilized bentonite presents a shift of maximum decomposition rate towards higher temperatures (220÷225°C for PB/ MA-VA nanocomposites and 220 ÷365 °C for PB/ MA-MMA nanocomposites), on the second step of decomposition, similar to maleic copolymers (Table 3). An increase of maleic copolymer concentration (from 50% to approximately 200% reported to bentonite) reflects into an increase of weight loss at the temperature range of 200 ÷500 °C. The obtained results show the intercalation of maleic copolymers between the silicate layers. Thermal stability of obtained binary nanocomposites correlates with the degree of interaction between silicate and copolymer. We notice that in the case of PB/ MA-MMA nanocomposites the best thermal stability is obtained at 1:1 ratio while the best thermal stability for the PB/ MA-VA nanocomposites is obtained at 2:1 ratio. These results are in accordance with X-ray diffraction results.

Collagen - Modified Layered Silicate Biomaterials for Regenerative Medicine of Bone Tissue 139

II 200÷ 400 °C

PB 8.3/ 74 - - 1.4 MA-MMA 4.9 84.3/ 321 8.1 2.7 PB/ MA-MMA (1:1) 10.7 38.5/ 367 6.7 1.6 PB/ MA-MMA (1:2) 10.4 52.1/ 365 8.7 1.3 PB/ MA-MMA (2:1) 8.4 22.1/ 218 7.5 5.4 MA-VA 7.6 61.3/ 258 27.9 0.9 PB/ MA-VA (1:1) 7.4 35.6/ 223 14.4 3.3 PB/ MA-VA (1:2) 7.1 27.2/ 220 8.2 6 PB/ MA-VA (2:1) 7.4 25.2/ 226 4.5 7.9

Table 3. TGA results for PB/ Maleic copolymer binary nanocomposites as compared to TGA

The layered silicate can improve thermal stability of collagen and depending on the collagen morphology (gel or colloidal solutions) and on the method used for silicate dispersion, a variety of lamellar structures and morphologies – intercalated or exfoliated- can be obtained

It is well known that the interaction of collagen with maleic copolymers takes place prevalently through weak forces and, to a little extent, through electrostatic interaction between the carboxylic groups of copolymer and the aminic groups of collagen

Maleic anhydride functionalized polymers ensure organosilicate dispersion and exfoliation into the polymer matrix (i.e. polypropylene) due to the interaction that may occur between maleic anhydride and hydroxyl groups on the silicate surface (Faisant et al., 1998; Vuluga et

Considering the results of these studies, it is reasonable to believe that a high adhesion to the collagen/modified layered silicate interface due to the interaction between the components leads to a uniform dispersion of modified layered silicate into collagen matrices

A dispersion of 0.5% of layered silicate modified with maleic copolymer in water: alcohol (1:1) mixture is added, when stirring, at room temperature, into a solution of collagen with 1.2% dried substance to obtain ternary nanocomposites. The different types of nanocomposites obtained in liquid form (which contain 5-10% of MMT reported to collagen dried substance) are crosslinked with 0.1% formaldehide, reported to collagen dried substance and are dried by lyophilization to obtain spongious matrices. The freeze-drying process is an advantageous conditioning technique which consists of drying frozen samples by ice sublimation into vacuum. To maintain the triple helix conformation of collagen molecules we use the following process: the liquid ternary nanocomposites are cast in stainless steell plates, cooling to -40 0C (4 hours), kept up for 4 hours, then freeze-dried at -

I 20 ÷200 °C

Trdmax Temperature at the maximum rate on the first decomposition step

**4.2 Collagen/ layered silicate/ maleic copolymer nanocomposites** 

results for maleic copolymers (MA-MMA and MA-VA).

(Anghelescu-Dogaru et al., 2004; Chitanu et al., 2002).

Weight loss on the each decomposition step (%)/ Trdmax (°C)

III 400÷ 550 °C

IV 550 ÷700 °C

Sample

(Vuluga et al., 2007).

al., 2008b; Utracki, 2002).

and to improved properties.

Fig. 7. X-ray diffractions for PB/ MA-MMA nanocomposites at different ratios.

Fig. 8. X-ray diffractions for PB/ MA-VA nanocomposites at different ratios.


Trdmax Temperature at the maximum rate on the first decomposition step

138 Materials Science and Technology

2 θ (°)

2 θ (°)

Fig. 8. X-ray diffractions for PB/ MA-VA nanocomposites at different ratios.

Fig. 7. X-ray diffractions for PB/ MA-MMA nanocomposites at different ratios.

Table 3. TGA results for PB/ Maleic copolymer binary nanocomposites as compared to TGA results for maleic copolymers (MA-MMA and MA-VA).

#### **4.2 Collagen/ layered silicate/ maleic copolymer nanocomposites**

The layered silicate can improve thermal stability of collagen and depending on the collagen morphology (gel or colloidal solutions) and on the method used for silicate dispersion, a variety of lamellar structures and morphologies – intercalated or exfoliated- can be obtained (Vuluga et al., 2007).

It is well known that the interaction of collagen with maleic copolymers takes place prevalently through weak forces and, to a little extent, through electrostatic interaction between the carboxylic groups of copolymer and the aminic groups of collagen (Anghelescu-Dogaru et al., 2004; Chitanu et al., 2002).

Maleic anhydride functionalized polymers ensure organosilicate dispersion and exfoliation into the polymer matrix (i.e. polypropylene) due to the interaction that may occur between maleic anhydride and hydroxyl groups on the silicate surface (Faisant et al., 1998; Vuluga et al., 2008b; Utracki, 2002).

Considering the results of these studies, it is reasonable to believe that a high adhesion to the collagen/modified layered silicate interface due to the interaction between the components leads to a uniform dispersion of modified layered silicate into collagen matrices and to improved properties.

A dispersion of 0.5% of layered silicate modified with maleic copolymer in water: alcohol (1:1) mixture is added, when stirring, at room temperature, into a solution of collagen with 1.2% dried substance to obtain ternary nanocomposites. The different types of nanocomposites obtained in liquid form (which contain 5-10% of MMT reported to collagen dried substance) are crosslinked with 0.1% formaldehide, reported to collagen dried substance and are dried by lyophilization to obtain spongious matrices. The freeze-drying process is an advantageous conditioning technique which consists of drying frozen samples by ice sublimation into vacuum. To maintain the triple helix conformation of collagen molecules we use the following process: the liquid ternary nanocomposites are cast in stainless steell plates, cooling to -40 0C (4 hours), kept up for 4 hours, then freeze-dried at -

Collagen - Modified Layered Silicate Biomaterials for Regenerative Medicine of Bone Tissue 141

Collagen Matrix pH 7.2 1.6 14.3/ 147 50.2/ 337 33.9/ 600

nanocomposite matrix pH 3 16.3 13.1/ 171 40.8/ 344 29.8/ 588

nanocomposite matrix pH 3 15.4 11.6/ 173 48.9/ 349 24.1/ 585

nanocomposite matrix pH 7 23.4 9.1/ 87; 172 41.6/ 351 25.2/ 586

I 0 ÷250 °C

Weight loss on each decomposition step (%)/Tvmax (°C)

> II 250 ÷500 °C

<sup>207</sup>39.3/ 345 25.2/ 605

III 500 ÷700 °C

(%)

Sample Residue at 700 °C,

nanocomposite matrix pH 7 18.9 16.3/ 171;

Table 4. Thermal stability for the ternary nanocomposite matrices.

a) b)

Fig. 10. Morphologic structure of (a) CG/ PB/ MA-MMA nanocomposites pH 7, compared

Tvmax, temperature at maximum rate of decomposition, (°C)

CG/ PB/ MA-VA

CG/ PB/ MA-VA

CG/ PB/ MA-MMA

CG/ PB/ MA-MMA

to (b) collagen morphology.

400C and 0.12 mbar for 10 hours, subsequently heated to +20 0C at a rate of 3 0C/hour (20 hours) at 0.12 mbar, heated again (6 hours) to 30 0C at a rate of 2 0C/hour and finally freezedried at the same temperature at 0.01 mbar for 4 hours, using the Christ Model Delta 2–24 LSC freeze-dryer (Germany). During the 48 hours of freeze-drying we obtain: CG/ PB/MA-VA and CG/ PB/MA-MMA spongious matrices.

We study both the influence of binary nanocomposite type and of the collagen solution pH upon ternary nanocomposite properties. Although the normal human body pH is 7.2 we still study the nanocomposite properties at an acidic pH of 3 knowing that in acidic environment collagen molecules are positively charged by the amine groups thus being able to interact more easily with the negatively charged layered silicates.

Both ternary nanocomposites (CG/PB/MA-MMA and CG/PB/MA-VA) present a partially exfoliated lamellar structure (Fig. 9). In the nanocomposites with hydrophobic comonomer (MMA) the degree of interaction is stronger at basic pH, thus we obtain partially exfoliated nanostructured biomaterials. When we use hydrophilic comonomer (VA), the degree of interaction is stronger at acidic pH. For biocompatibility tests, we select the nanocomposite with hydrophobic comonomer. The strong degree of interaction for the hydrophobic comonomer reflects itself in an improved thermal stability (Table 4).

Fig. 9. X-ray diffraction patterns for ternary nanocomposites with hydrophilic (a) or hydrophobic (b) comonomer, at acidic and basic pH.

As compared to collagen, the CG/ PB/MA-MMA nanocomposites are more thermally stable. Thermal stability for the ternary nanocomposites is higher at basic pH and with hydrophobic maleic comonomer (Table 4).

The nanocomposites CG/PB/MA-MMA (pH 7) present a spongious structure with interconnected macro, micro and nano sized pores, with nanostructured regions (Fig. 10 a). Collagen morphologic structure presented in secondary electron images (SEI) shows regions with large communicating pores as well as interconnected ones. We also notice the morphologic homogeneity of the collagen fibers (Fig. 10 b). Even though the amount of maleic copolymer modified layered silicate in ternary nanocomposites is less than the amount of collagen, a compact fibrous structure can be observed, which can be the reason of an increase of thermal stability.


Tvmax, temperature at maximum rate of decomposition, (°C)

140 Materials Science and Technology

400C and 0.12 mbar for 10 hours, subsequently heated to +20 0C at a rate of 3 0C/hour (20 hours) at 0.12 mbar, heated again (6 hours) to 30 0C at a rate of 2 0C/hour and finally freezedried at the same temperature at 0.01 mbar for 4 hours, using the Christ Model Delta 2–24 LSC freeze-dryer (Germany). During the 48 hours of freeze-drying we obtain: CG/ PB/MA-

We study both the influence of binary nanocomposite type and of the collagen solution pH upon ternary nanocomposite properties. Although the normal human body pH is 7.2 we still study the nanocomposite properties at an acidic pH of 3 knowing that in acidic environment collagen molecules are positively charged by the amine groups thus being able

Both ternary nanocomposites (CG/PB/MA-MMA and CG/PB/MA-VA) present a partially exfoliated lamellar structure (Fig. 9). In the nanocomposites with hydrophobic comonomer (MMA) the degree of interaction is stronger at basic pH, thus we obtain partially exfoliated nanostructured biomaterials. When we use hydrophilic comonomer (VA), the degree of interaction is stronger at acidic pH. For biocompatibility tests, we select the nanocomposite with hydrophobic comonomer. The strong degree of interaction for the hydrophobic

a) b)

As compared to collagen, the CG/ PB/MA-MMA nanocomposites are more thermally stable. Thermal stability for the ternary nanocomposites is higher at basic pH and with

The nanocomposites CG/PB/MA-MMA (pH 7) present a spongious structure with interconnected macro, micro and nano sized pores, with nanostructured regions (Fig. 10 a). Collagen morphologic structure presented in secondary electron images (SEI) shows regions with large communicating pores as well as interconnected ones. We also notice the morphologic homogeneity of the collagen fibers (Fig. 10 b). Even though the amount of maleic copolymer modified layered silicate in ternary nanocomposites is less than the amount of collagen, a compact fibrous structure can be observed, which can be the reason of

Fig. 9. X-ray diffraction patterns for ternary nanocomposites with hydrophilic (a) or

hydrophobic (b) comonomer, at acidic and basic pH.

hydrophobic maleic comonomer (Table 4).

an increase of thermal stability.

VA and CG/ PB/MA-MMA spongious matrices.

to interact more easily with the negatively charged layered silicates.

comonomer reflects itself in an improved thermal stability (Table 4).

Table 4. Thermal stability for the ternary nanocomposite matrices.

a) b)

Fig. 10. Morphologic structure of (a) CG/ PB/ MA-MMA nanocomposites pH 7, compared to (b) collagen morphology.

Collagen - Modified Layered Silicate Biomaterials for Regenerative Medicine of Bone Tissue 143

The key results consist in obtaining new types of collagen nanostructured biomaterials in the form of spongious, microporous matrices, nontoxic and biocompatible with osteoblast cells. The preparation of the new nanocomposites is based on the use of two natural components: collagen, which contributes to bone regeneration and natural layered silicate (montmorillonite) which improves the thermal resistance of collagen, able to release in time an active substance at low and controlled concentrations. Maleic copolymers favour the silicate dispersion in the matrix of collagen and the obtaining of intercalated partially exfoliated nanocomposites with disordered lamellar structure. The nanocomposites in form of microporous matrix (scaffold) obtained by dispersion of binary nanocomposites in collagen gel have a spongy structure which contains macro and micro interconnected nanopores, similar with extra cellular matrix, which allows the penetration of the physiological fluids, necessary for the growth of cells. In comparison with collagen matrix,

The financial support of MATNANTECH Program of the Romanian Ministry of Education and Research, by means of CEEX project no. 16/2005-2007, to achieve this contribution is

The authors are gratefully acknowledged for their collaboration with C. Radovici for XRD, C. Nistor for DLS and S. Serban for TGA from National Research and Development Institute for Chemistry and Petrochemistry-ICECHIM, Bucharest and to "Petru Poni" Institute of Macromolecular Chemistry, Romanian Academy, Iasi team which provided maleic

Al-Marhoun, M.A. & Rahman, S.S. (1988). Optimizing the Properties of Water-Based

Albrektsson, T. & Johansson, C. (2001). Osteoinduction, Osteoconduction and

Albu, M.G.; Ghica, M.V.; Giurginca, M.; Trandafir, V.; Popa, L. & Cotrut, C. (2009). Spectral

Albu, M.G. (2011). *Collagen gels and matrices for biomedical applications,* LAP LAMBERT

Allen, T.M. & Cullis, P.R. (2004). Drug Delivery Systems: Entering the Mainstream. *Science*,

Vol. 303, No. 5665, (March 2004), pp. (1818-1822), ISSN 1095-9203

Polymer Drilling Fluids for Penetrating Formations with Electrolyte Influx. *Erdoel, Erdgas, Kohle*, Vol. 104, No. 7-8, (September 1988), pp. (318-323), ISSN 0179-3187 Albrektsson, T. (1995). Principles of Osseointegration, In: *Color Atlas and text of Dental and* 

*maxillofacial implantology*, J.A. Hobkirk, K. Watson (Ed.), pp. (9–19), Mosby-Wolfe,

Osseointegration. *European Spine Journal*, Vol. 10, No. 2, (October 2001), pp. (96–

characteristics and antioxidant properties of tannic acid immobilized on collagen drug-delivery systems. *Revista de Chimie – Bucharest*, Vol.60, No.7, (July 2009), pp.

Academic Publishing GmbH & Co. KG, ISBN 978-3-8443-3057-1, Saarbrucken,

CG/ PB-MA-MMA nanocomposites have improved thermal stability.

ISBN 9780723417866, London, United Kingdom

101), ISSN 1432-0932

666-672, ISSN 0034-7752

Germany

**5. Conclusions** 

**6. Acknowledgment** 

gratefully acknowledged.

copolymers.

**7. References** 

The spongious matrix of CG/ PB/ MA-MMA nanocomposite was *in vitro* tested on osteoblast cell cultures. *In vitro* test results are presented in Table 5. After 24 and 72 hours since hatching, the cells proliferate on matrix surface, the viability being of 97%, very close to the reference value (control sample). These results are also supported by microscopy observation of cellular density (Fig. 11). The collagen/layered silicate/MA-MMA ternary nanocomposite does not present any citotoxic response and the cells present normal phenotype.


Table 5. Proliferation and osteoblast cells viability hatched on matrix of CG/ PB/ MA-MMA nanocomposites.

After 24 hours since hatching

After 72 hours from hatching

Fig. 11. Microscopic analysis of osteoblast culture samples sowed on CG/ PB/ MA-MMA ternary nanocomposite substrate after 24 and 72 hours since hatching.
