**2. Materials and characterization methods**

#### **2.1 Materials**

#### **2.1.1 Collagen gel**

Collagen gel (CG) is a polydisperse colloidal system which consists in molecules with native triple helix conformation and it is extensively used to obtain collagen-based biomaterials. Collagen gel is defined as a system with intermediate properties which vary between a liquid and a solid. It is characterized by structural viscosity, pseudoplastic behaviour and its average molecular weight is about 300000 Da, a value corresponding to the native collagen molecule weight (Trandafir et al., 2007).

Mild chemical and/or enzymatic treatments allow the extraction of collagen from calf hide into aqueous medium with preservation of triple helical structure of molecules, of microfibrils and fibrils. The collagen obtained is a gel, type I fibrillar collagen.

Type I collagen gel at a concentration of 2.3% and pH = 3 is obtained from calf hide by the current technology used in the Collagen Department in Leather and Footwear Research Institute, Bucharest, Romania (Albu, 2011; Budrugeac et al., 2003; Siapi et al., 2005). The obtained gel is physically-chemically characterized by 2.17% protein substance and 0.13% ash contents. The circular dichroism results show the technology used allows obtaining type I collagen with a high degree of purity and with triple helical structure (Albu, 2011). Absence of fat from this gel shows the high degree of purity. We use 1.2% gels to obtain collagen biomaterials.

### **2.1.2 Purified bentonite (natural layered silicate)**

Purified bentonite (natural layered silicate) characterized by 82% montmorillonite (MMT), by basal spacing, d001 = 14.7 Å, by 8% weight loss in the range of temperature 20 ÷ 200 °C (TGA) and by R1,0=0%; R0,5=0,2%; R0,063=65,8%; R0,04=97,1%, R0,025=100%, residue on sieve is obtained by purification of Romanian sodium bentonite.

### **2.1.3 Maleic copolymers**

130 Materials Science and Technology

cheap and available. From the synthetic polymers used in biomedicine, the copolymers of maleic anhydride (MA) were selected, because of their "drug-delivery" properties*.* In the field of medical and pharmaceutical applications, MA copolymers are used as components of some polymer - drug systems with controlled release (Gupta et al., 2002). Using MA copolymers for pharmaceutical and biomedical applications has advantages related to the structure, activity and their properties. MA copolymers have, in general, a relatively well characterized and reproducible alternating structure, hydrophilic or hydrophobic character and charge density can be varied by appropriate choice of comonomer (comonomers) (Edlund & Albertsson, 2002)*.* Although polyanhydrides are best to the drug delivery application, they were not used in orthopedic implantation due to low load bearing and mechanical properties. However, MA copolymers with considerable enhanced mechanical properties were specially designed for the orthopedic applications such as poly (anhydridee-imides) which can be used as scaffold for bone tissue engineering application (Sabir et al.,

In this chapter, we intend to present the obtaining and properties of new types of nanocomposites based on natural polymer (collagen) and bioactive compounds (natural layered silicate modified with maleic copolymers) which can be used as biomaterial, scaffold

Binary nanocomposites of layered silicate/maleic copolymers can contain different bioactive substances which can be released in time and at low and controlled concentrations (bone growth factors, titania or carbon aerogels, hydrolyzed collagen, anticancer compounds,

modification of the layered silicate surface (organophilization) with maleic

 uniform dispersion of binary nanocomposites in collagen gel thus obtaining ternary nanocomposites from which, by lyophilization, spongy matrices can be

The binary nanocomposites have high thermal stability which can allow the usage of some bioactive substances activated by local heating. These nanocomposites are compatible with collagen gel in such away biocompatible ternary nanocomposites matrices useful for

Collagen gel (CG) is a polydisperse colloidal system which consists in molecules with native triple helix conformation and it is extensively used to obtain collagen-based biomaterials. Collagen gel is defined as a system with intermediate properties which vary between a liquid and a solid. It is characterized by structural viscosity, pseudoplastic behaviour and its average molecular weight is about 300000 Da, a value corresponding to

Obtaining of collagen/modified layered silicate nanocomposites implies: layered silicate purification for usage in the biomedical field;

copolymers thus obtaining binary nanocomposites;

regenerative medicine of bone tissue can be obtained.

the native collagen molecule weight (Trandafir et al., 2007).

**2. Materials and characterization methods** 

2009).

for bone tissue regeneration.

antimicrobial antioxidants, etc.).

obtained.

**2.1 Materials** 

**2.1.1 Collagen gel** 

Maleic copolymers having variable hydrophobicity (maleic anhydride copolymers with a hydrophilic comonomer - vinyl acetate and a hydrophobic comonomer - methyl methacrylate), are obtained in anhydrous form by "Petru Poni" Institute of Macromolecular Chemistry, Romanian Academy, Iasi.

The monomers, catalysts, and solvents are carefully purified before usage, according to already known methods (Caze & Loucheux, 1975 as cited in Chitanu et al., 2006; Riddick & Burger, 1970 as cited in Chitanu et al., 2006).

The MA-VA copolymers of maleic anhydride (MA) with vinyl acetate (VA), (Chitanu et al., 2006) and MA-MMA copolymers with methyl methacrylate (MMA), (Chitanu et al., 2007) are synthesized by free radical polymerization, purified by extraction and washed with diethyl ether. The composition is estimated by conductometric titration with aqueous 0.1N NaOH in 1:1 acetone-water mixture and the molecular weight (Mw) by viscosimetric measurements in acetone at 30 °C.

The chemical structures of maleic copolymers used are presented in Fig. 1 and 2.

Fig. 1. Chemical structure of MA-VA copolymer.

Collagen - Modified Layered Silicate Biomaterials for Regenerative Medicine of Bone Tissue 133

samples are studied at different magnitude orders using a HITACHI scanning electron microscope model S-2600N equipped with an energy dispersive X-ray spectrometer

Using a Zeta Seizer MALVERN UK, the average diameter of submicron particles is

Thermal behavior of the obtained materials is analyzed by determining the sample weight loss when heated at a constant rate (TGA), depending on temperature. By differential thermal analyses (DTA) we determine the temperature at which we get the highest decomposition rate. Thermal tests are performed on THERMAL ANALYST DUPONT 2100

Biocompatibility properties are evaluated on already obtained binary/ternary systems using *in vitro* tests. Biocomposite matrices are cut into small samples (1 cm2) and are sterilized in ultraviolete light on the both sides. The samples are sown with osteoblast cells from G 292 cellular line at an initially cellular density of 3.5 x 105 cells/plate. The obtained cultures are hatched at 37 °C in moist atmosphere with 5% CO2, then monitored from cytomorphologically point of view at 24 and 72 hours and from cellular viability point of view after 72 hours since hatching. After 24 hours, the matrices are observed under a Nikon TS 100 microscope in phase contrast and pictures are taken using a Nikon Cooplix 4500 digital camera. After 72 hours, the cultures are stained with hypericin and pictures are

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

Purification is made by dispersing the bentonite in distilled water at 60 ÷90 °C. Quartz and

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

other hydrophobic impurities are separated by decantation.

**2.2.3 Dynamic diffusion of light with laser source (DLS)** 

determined in aqueous suspension ranging 3 nm ÷1 μm.

(EDAX).

using:

taken.

**2.2.4 Thermal analyses** 

heating rate: 20 °C/min;

**3. Bentonite purification** 

process (Carrado, 2006).

**2.3 Biocompatibility** 

temperature range: 0 ÷ 700 °C.

in air with the flow rate of 50 cm3/min;

Fig. 2. Chemical structure of MA-MMA copolymer.

The characteristics of MA-MMA maleic copolymers with hydrophobic comonomer and of MA-VA maleic copolymers with hydrophilic comonomer are presented in Table 1.


Table 1. The characteristics of maleic copolymers.

#### **2.2 Methods of physical-chemical and structural characterization**

Physical-chemical and structural properties of purified layered silicate, of binary nanocomposites based on layered silicate/maleic copolymers and of ternary nanocomposites of collagen/layered silicate/maleic copolymers are determined by using different techniques described below.

#### **2.2.1 X-ray diffraction**

X-ray diffraction technique is used to determine the basal spacing. Difractograms are recorded on automatic system using a DRON-20 difractometer, with a horizontal goniometer and a scintillation counter. We used as radiation source, CoK (λ = 1.7902 Å) filtered with Fe in order to remove kβ component from the Bragg-Brentano system (in reflexion mode). *Basal spacing* is determined using Bragg equation1:

$$\mathbf{n}\lambda = \mathbf{2d}\sin\theta, \mathbf{d} = \frac{\mathbf{n}\lambda}{2\sin\theta} \tag{1}$$

where *n* represents the reflexion order, *λ* is the wavelength of X-rays, *θ* is the diffraction angle, *d* is the distance between the planes of crystalline network which produces the diffraction.

#### **2.2.2 Scanning electron microscopy (SEM)**

Nanocomposite particles shape and dimension are observed using a scanning electron microscope and microanalyzed with an energy dispersive X-ray spectrometer (EDAX). The samples are studied at different magnitude orders using a HITACHI scanning electron microscope model S-2600N equipped with an energy dispersive X-ray spectrometer (EDAX).
