**3. Bentonite purification**

Knowing that the bentonite may contain different percentages (5÷40%) of quartz and other impurities, it is necessary to purify the natural layered silicate (bentonite). These impurities must be removed because in the surface modification process, in order to ensure the compatibility between the silicate and polymer matrix, they act as a sterile, hindering the process (Carrado, 2006).

Purification is made by dispersing the bentonite in distilled water at 60 ÷90 °C. Quartz and other hydrophobic impurities are separated by decantation.

Small amounts of bentonite (approximately 5 g) are added into 100 ml of hot distilled water (60÷90 °C) under mixing conditions. The mixing is followed in a ball mill for 2 hours until the suspension becomes homogenous. The obtained mixture is allowed to settle for 72 hours, then the upper layer is separated (the purified suspension) and dried for 24 hours at

Collagen - Modified Layered Silicate Biomaterials for Regenerative Medicine of Bone Tissue 135

 Fig. 4. Secondary electron images (SEI) and distribution of X-rays for purified bentonite.

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,

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

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.

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

Ficai et al., 2010b, Lungu et al., 2007, Titorencu et al., 2010).

ulcerous wound (Trandafir et al., 2007).

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

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

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 et al., 2008b).

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 impurities concentration and the increase of the amount of montmorillonite.


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

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 relatively uniformly dispersed.

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

Fig. 4. Secondary electron images (SEI) and distribution of X-rays for purified bentonite.
