**1. Introduction**

The interdisciplinary field of polymer nanocomposite biomaterials brings together researchers from polymer science, biology, materials and biomedical engineering, chemistry and physics. In order to understand the need of biomaterials in our life, first we need to define and understand the specific terms that link these fields together. It is obvious that, due to the fact that the field of biomaterials is quite a new interdisciplinary one, the same term can be found with several explanations.

"The application of the principles of natural sciences, especially of biology, biochemistry and physiology to clinical medicine" was defined as **biomedicine** (Miller & O'Toole, 2005, as cited in Medical Dictionary, ndc; Picket, 2000, as cited in Medical Dictionary, ndc).

Collaboration between fields leads to the generation and use of new terminology and definitions that are used across each discipline in part. For example, the traditional definition of biomaterials has changed in time (Wu et al., 2010) as these materials find their use in a variety of medical and nonmedical technologies that are inspired by biology (Huebsch & Mooney, 2009; Williams, 2009; Wu et al., 2010). In 1987, The European Society of Biomaterials (Williams, 1987) defines the biomaterial as "a non viable material used in a medical device, intended to interact with biological systems." Later on, in 1999 (Williams, 1999), in the dictionary of biomaterial science, the definition of biomaterial was changed, saying that it represents "a material intended to interface with biological systems to evaluate, treat, augment or replace any tissue, organ or function of the body" (Williams,

Collagen - Modified Layered Silicate Biomaterials for Regenerative Medicine of Bone Tissue 127

These requirements derive from the bone complex structure and properties. Bone is a complex and dynamic living tissue, which from morphological point of view can be divided into two types: cortical or compact bone and trabecular or cancellous or spongy bone. The mass of the skeleton is composed of 80% compact bones and 20% spongy bones. Cortical and trabecular bones contain the same cells and extracellular matrix (ECM) components, except that they are organized in a different way. The trabecular bone consists of a porous matrix with interconnected columns filled with bone marrow and is being responsible for metabolic functions of the bone, while the cortical bone contains fewer spaces, forms the external layer of all bones and provides them protection and load-bearing capabilities

Extracellular matrix is the most abundant component in bone tissue and is composed from 30% of an organic phase of collagen and of other proteins and from 70% of an inorganic phase of calcium phosphate. The organic phase provides elasticity and strength, while the inorganic phase gives hardness and load-bearing capabilities (Mundy et al. 2003, as cited in

Remodelling is a physiological process in which the bones are being built, resorbed and then rebuilt again and that process is carried out and carefully controlled by a variety of cell types. Osteoblasts synthesize and mineralize the bone matrix which is maintained by

A biomaterial to be used as scaffold in tissue engineering must satisfy a number of requirements. These requirements include biocompatibility, biodegradation to non-toxic products within the period required for the application, processability to complicated shapes with appropriate porosity, ability to support cell growth and proliferation, ability to incorporate cells, growth factors, appropriate mechanical properties, as well as maintaining mechanical strength during the tissue regeneration process. All these requirements need to be fulfilled by implanted materials in order to achieve "biointergration". However, the term of biointegration is not completely accepted by researchers in the medical field (Szmukler-Moncler & Dubruille, 1990). Dubruille et al. did not agree to define biointegration as "describing the direct biochemical bonding of bone to an implant surface". For this situation they propose a new term "osseointegration" since the "biointegration" does not refer only to the integration with bone. Even though, we will find that biointegration term is still used and universally accepted as referring to the bioactivity of implanted material with bone

Biocompatibility was defined as being "the property of being biologically compatible by not producing a toxic, injurious, or immunological response in living tissue" (Picket, 2000, as

In biology, the term "cell growth" is used in two different ways. In the context of reproduction of living cells the phrase "cell growth" is used for the idea of cell reproduction meaning growth in cell numbers. During cell reproduction the "parental" cell divides into two "daughter" cells. In other contexts, "cell growth" refers to increases in cell size (Conlon &

1. The biocompatibility between the material and the surrounding environment. 2. The mechanical and physical properties necessary to achieve the desired function. 3. The relative ease of fabrication and supply of the required components (Hoeppner &

Chandrasekaran, 1994).

(Baron, 2003, as cited in Wilson, 2011).

tissue (Blokhuis et al., 2000).

cited in Medical Dictionary, nda).

Wilson, 2011; Robey & Boskey, 2008, as cited in Wilson, 2011).

osteocytes, and as required, is resorbed by osteoclasts (Wilson, 2011).

2009). In a 2003 medical encyclopedia (Miller & O'Toole, 2005 as cited in Medical Dictionary, ndb) the authors combine the 1999 and 2002 definitions saying that the biomaterial can be defined as "any substance (other than a drug), synthetic or natural, that can be used as a system or part of a system that treats, augments, or replaces any tissue, organ, or function of the body; especially, material suitable for use in prostheses that will be in contact with living tissue". In 2005, modern medicine (Segen, 2005 as cited in Medical Dictionary, ndb) gives two definitions for biomaterial as being - "1. any synthetic material or device – e.g. implant or prosthesis - intended to treat, enhance or replace an aging or malfunctioning–or cosmetically unacceptable — native tissue, organ or function in the body, bioengineering, breast implants, hybrid artificial pancreas, Shiley valve, teflon, total hip replacement and 2. a biomaterial used for its structural, not biological properties – e.g., collagen in cosmetics, carbohydrates modified by biotechnology to be used as lubricants for biomedical applications or as bulking agents in food industry." The latest definition in 2009 (Williams, 2009), which includes all the previous ideas, states that "A biomaterial is a substance that has been engineered to take a form which, alone or as part of a complex system, is used to direct, by control of interactions with components of living systems, the course of any therapeutic or diagnostic procedure, in human or veterinary medicine." Facing this changes in time, makes it difficult to classify polymer nanocomposite biomaterials into materials which are intended for medical devices and which implies their use for bio-technological purposes (e.g., renewable resources) (Wu et al., 2010). Polymeric biomaterials are developed in different shapes and for different purposes in order to substitute and repair biological tissues (Hench & Polak, 2002; Ma & Elisseeff, 2005, as cited in Wu et al., 2010). New attempts to design self-assembled and smart nanocomposite biomaterials are conducted to fulfil some external requirements such as optic, temperature, mechanic, electric and magnetic fields (Hirst et al., 2008; Peppas et al., 2006; Stuart et al. 2010). These requirements are very important when designing smart implants and drug delivery systems as well as new biotechnologies including biosensors, *in vitro* diagnostics, cell culture matrices, contrast agents and bioassays (Hirst et al., 2008; Stuart et al. 2010; Wu et al., 2010).

Initially the implants were intended mainly to replace affected (i.e., broken bones, heart, vein, liver, pancrease) or malfunctioning body parts which people cannot live without. Subsequently, the industry of implants developed fast and now there are implants which are used for reconstructing and aesthetic purposes such as breast implants, bone facial implants and skin implants. According to the type of replaced tissue, they are divided into hard and soft implants. Hard tissue implants are usually made of metals or ceramics (bone implants, dental sealant, and joints) while soft tissue implants are made of polymers (blood vessels, skin, plastic surgery).

Essential components of modern medicine have become orthopaedic implants (represented by metallic implants as well as bone cements) (Widmer, 2001).

In recent years, orthopaedic implants and prostheses have imposed an increasing demand on the materials used. The more complex the function of the implant becomes, the more complex the requirements of the constructional material are. The success or failure of any device is often dependent on the choice of material as it is on the configurational and functional design.

The requirements that modern bone implants are expected to meet can be divided into three groups:

2009). In a 2003 medical encyclopedia (Miller & O'Toole, 2005 as cited in Medical Dictionary, ndb) the authors combine the 1999 and 2002 definitions saying that the biomaterial can be defined as "any substance (other than a drug), synthetic or natural, that can be used as a system or part of a system that treats, augments, or replaces any tissue, organ, or function of the body; especially, material suitable for use in prostheses that will be in contact with living tissue". In 2005, modern medicine (Segen, 2005 as cited in Medical Dictionary, ndb) gives two definitions for biomaterial as being - "1. any synthetic material or device – e.g. implant or prosthesis - intended to treat, enhance or replace an aging or malfunctioning–or cosmetically unacceptable — native tissue, organ or function in the body, bioengineering, breast implants, hybrid artificial pancreas, Shiley valve, teflon, total hip replacement and 2. a biomaterial used for its structural, not biological properties – e.g., collagen in cosmetics, carbohydrates modified by biotechnology to be used as lubricants for biomedical applications or as bulking agents in food industry." The latest definition in 2009 (Williams, 2009), which includes all the previous ideas, states that "A biomaterial is a substance that has been engineered to take a form which, alone or as part of a complex system, is used to direct, by control of interactions with components of living systems, the course of any therapeutic or diagnostic procedure, in human or veterinary medicine." Facing this changes in time, makes it difficult to classify polymer nanocomposite biomaterials into materials which are intended for medical devices and which implies their use for bio-technological purposes (e.g., renewable resources) (Wu et al., 2010). Polymeric biomaterials are developed in different shapes and for different purposes in order to substitute and repair biological tissues (Hench & Polak, 2002; Ma & Elisseeff, 2005, as cited in Wu et al., 2010). New attempts to design self-assembled and smart nanocomposite biomaterials are conducted to fulfil some external requirements such as optic, temperature, mechanic, electric and magnetic fields (Hirst et al., 2008; Peppas et al., 2006; Stuart et al. 2010). These requirements are very important when designing smart implants and drug delivery systems as well as new biotechnologies including biosensors, *in vitro* diagnostics, cell culture matrices, contrast agents

and bioassays (Hirst et al., 2008; Stuart et al. 2010; Wu et al., 2010).

by metallic implants as well as bone cements) (Widmer, 2001).

vessels, skin, plastic surgery).

functional design.

groups:

Initially the implants were intended mainly to replace affected (i.e., broken bones, heart, vein, liver, pancrease) or malfunctioning body parts which people cannot live without. Subsequently, the industry of implants developed fast and now there are implants which are used for reconstructing and aesthetic purposes such as breast implants, bone facial implants and skin implants. According to the type of replaced tissue, they are divided into hard and soft implants. Hard tissue implants are usually made of metals or ceramics (bone implants, dental sealant, and joints) while soft tissue implants are made of polymers (blood

Essential components of modern medicine have become orthopaedic implants (represented

In recent years, orthopaedic implants and prostheses have imposed an increasing demand on the materials used. The more complex the function of the implant becomes, the more complex the requirements of the constructional material are. The success or failure of any device is often dependent on the choice of material as it is on the configurational and

The requirements that modern bone implants are expected to meet can be divided into three


These requirements derive from the bone complex structure and properties. Bone is a complex and dynamic living tissue, which from morphological point of view can be divided into two types: cortical or compact bone and trabecular or cancellous or spongy bone. The mass of the skeleton is composed of 80% compact bones and 20% spongy bones. Cortical and trabecular bones contain the same cells and extracellular matrix (ECM) components, except that they are organized in a different way. The trabecular bone consists of a porous matrix with interconnected columns filled with bone marrow and is being responsible for metabolic functions of the bone, while the cortical bone contains fewer spaces, forms the external layer of all bones and provides them protection and load-bearing capabilities (Baron, 2003, as cited in Wilson, 2011).

Extracellular matrix is the most abundant component in bone tissue and is composed from 30% of an organic phase of collagen and of other proteins and from 70% of an inorganic phase of calcium phosphate. The organic phase provides elasticity and strength, while the inorganic phase gives hardness and load-bearing capabilities (Mundy et al. 2003, as cited in Wilson, 2011; Robey & Boskey, 2008, as cited in Wilson, 2011).

Remodelling is a physiological process in which the bones are being built, resorbed and then rebuilt again and that process is carried out and carefully controlled by a variety of cell types. Osteoblasts synthesize and mineralize the bone matrix which is maintained by osteocytes, and as required, is resorbed by osteoclasts (Wilson, 2011).

A biomaterial to be used as scaffold in tissue engineering must satisfy a number of requirements. These requirements include biocompatibility, biodegradation to non-toxic products within the period required for the application, processability to complicated shapes with appropriate porosity, ability to support cell growth and proliferation, ability to incorporate cells, growth factors, appropriate mechanical properties, as well as maintaining mechanical strength during the tissue regeneration process. All these requirements need to be fulfilled by implanted materials in order to achieve "biointergration". However, the term of biointegration is not completely accepted by researchers in the medical field (Szmukler-Moncler & Dubruille, 1990). Dubruille et al. did not agree to define biointegration as "describing the direct biochemical bonding of bone to an implant surface". For this situation they propose a new term "osseointegration" since the "biointegration" does not refer only to the integration with bone. Even though, we will find that biointegration term is still used and universally accepted as referring to the bioactivity of implanted material with bone tissue (Blokhuis et al., 2000).

Biocompatibility was defined as being "the property of being biologically compatible by not producing a toxic, injurious, or immunological response in living tissue" (Picket, 2000, as cited in Medical Dictionary, nda).

In biology, the term "cell growth" is used in two different ways. In the context of reproduction of living cells the phrase "cell growth" is used for the idea of cell reproduction meaning growth in cell numbers. During cell reproduction the "parental" cell divides into two "daughter" cells. In other contexts, "cell growth" refers to increases in cell size (Conlon &

Collagen - Modified Layered Silicate Biomaterials for Regenerative Medicine of Bone Tissue 129

One of the mostly researched biomaterials used in various fields of medicine is natural polymer-collagen. Collagen is the most abundant protein in the body (skin, bones, teeth, tendons, cartilage, basement membrane, cornea, etc.). It can be used by different processing techniques in large various molecular structures (micro and nanostructures) as powder,

The use of collagen as biomaterial, biocompatible and bioresorbable for the connective

To use collagen as scaffold in the recovery of bone tissue modifications in the structure and composition of matrix we need to obtain osteoinductive and osteoconductive effect. This can be achieved by the formation of biocomposites with SiO2, TiO2, clay, hydroxyapatite, etc (Christiansen et al., 2011; Vuluga et al., 2008a). Osteoinduction is the effect that appears in bone healing and involves the recruitment and stimulation of immature cells to develop into preosteoblast cells. Osteoconduction refers to bone growth on an implanted material, including bone conduction on its surface or down into pores, channels or pipes (Albrektsson & Johansson, 2001). Osteoconductive effect depends not only on biological factors, but also on the response to a foreign material. Bone conduction is not possible on certain materials, like some metals (Cu, Ag) (Albrektsson, 1995, as cited in Albrektsson & Johansson, 2001), while osteoinductive effect was observed on a large number of biomaterials (Barradas et al.,

The layered silicate improves the thermal and mechanical properties of collagen and has a biostimulative effect on cellular metabolism. The structure of natural silicate would be like the structure of the living tissue because of the ionic exchange properties in aqueous medium just like the physiological phenomena in the human body where permanent information exchanges are present. Silicates surface can be modified with different bioactive substances and obtained nanohybrids can be dispersed in a collagen matrix (Potarniche et al., 2010; Potarniche et al. 2011). Depending on the concentration and the method of the silicate incorporation in the collagen protein, nanostructured systems with different morphologies, capable to control release of active principles (antibiotics, anti-inflammatory, enzymes, etc.) can be obtained. These types of nanocomposites can be used in regenerative

Polymer/inorganic nanocomposites are materials with unique properties which have found applications in large fields of activity: aircraft industry, automobile, packaging, construction, electronics, electrical engineering and last but not least in medicine and pharmaceutical industry. To obtain nanocomposites hybrids, the clay minerals (hydrated layered silicates) are preferred due to their high mechanical and chemical strength and low cost. The biomedical properties of clays are known since antiquity, these being in principal antiseptic, bactericides, scar action, antitoxic, without microbial germs. The special surface physical and chemical properties, such as high specific surface, layer charge, swelling capacity, make clay and clay minerals to be useful for human health in both external and "per os" (internal) applications. Clays can be used as active principles or excipients in pharmaceutical formulas, cosmetic medicine, in treating intestinal disorders and as drugs, being good

In medicine, synthetic polymers are also frequently used, not because of their biological activity, but because they are relatively easy to obtain, they are inert to body fluids, they are

injectable solutions, films, membrane and matrices (sponges).

medicine of bone tissue (Christiansen et al., 2011).

adsorbents and mucostabilizers (Choy et al., 2007).

2011).

tissues prosthesis in which collagen is the basic protein is very well known.

Raff, 1999). The first definition of the cell growth is also attributed to the word "proliferation" which is defined as "to grow or multiply by rapidly producing new tissue, parts, cells, or offspring" (Picket, 2000, as cited in Medical Dictionary, ndd) when we refer only to cells. So, in material science in order to differentiate between the two concepts, we refer to cell growth when the size of the cell increases and use the term proliferation when the number of the cells increases.

Another important issue that needs to be considered, when obtaining an implant, is body response. Upon implantation, cell adhesion and fibrous tissue encapsulation takes place as a defensive response of the body (Recum & Strauss, 1998, as cited in Shin & Schoenfisch, 2006; Wilson & Gifford, 2005). The wound healing response initiates as an immune response. There were differentiated four distinct stages of the wound healing which are met starting after few seconds until complete healing: hemostasis, inflammation, repair, and encapsulation. Bacterial infection that can occur due to bacterial adhesion is a serious problem that affects implanted materials during healing or in the case of extended periods of time (Dankert et al., 1986, as cited in Shin & Schoenfisch, 2006; Sawan & Manivannan, 2000, as cited in Shin & Schoenfisch, 2006). In order to overcome this problem, strategies are being developed based on control release of an anticoagulant or antimicrobial agent from implanted material (Christiansen et al., 2011; Hendricks et al., 2000; Shin & Schoenfisch, 2006; Williams, 2009).

Drug delivery systems (DDS) are new types of drug carriers, designed to improve the pharmacological and therapeutic properties of administrated drugs (Allen & Cullis, 2004). Such systems are designed according to the type of administration. There are transdermal drug delivery systems (TDDS) used for external administration of drugs and liposomal and targeted drug delivery systems (LTDDS) for internal administration. TDDS present the advantages of avoiding hepatic first pass metabolism, of decreasing gastrointestinal effect and improving compliance, while LTDDS are used to reduce the dose and increase the activity of the drug at the precise site (Kshirsagar, 2000). DDS can be classified after their release mechanism into (Lager & Peppas, 1981):

	- a. Reservoirs (membrane devices)
	- b. Matrices (monolithic devices)
	- c. Bioerodible systems
	- d. Pendant Chain systems

The dose released can differ according to the type of the system used and can be: extended release, slow release and sustained release (Kshirsagar, 2000).

Generally, DDS present zero-order kinetics and they should be inert and biocompatible with the environment. As compared to the conventional way of drug administration, DDS have the advantages of being more comfortable for the user, easily administrated, possess a high drug concentration, have good mechanical strength, safe and free from leaks. They also have biocompatible, non-toxic, nonmutagenic, non-carcinogenic, non-teratogenic and nonimmunogenic properties (Lager & Peppas, 1981).

Raff, 1999). The first definition of the cell growth is also attributed to the word "proliferation" which is defined as "to grow or multiply by rapidly producing new tissue, parts, cells, or offspring" (Picket, 2000, as cited in Medical Dictionary, ndd) when we refer only to cells. So, in material science in order to differentiate between the two concepts, we refer to cell growth when the size of the cell increases and use the term proliferation when

Another important issue that needs to be considered, when obtaining an implant, is body response. Upon implantation, cell adhesion and fibrous tissue encapsulation takes place as a defensive response of the body (Recum & Strauss, 1998, as cited in Shin & Schoenfisch, 2006; Wilson & Gifford, 2005). The wound healing response initiates as an immune response. There were differentiated four distinct stages of the wound healing which are met starting after few seconds until complete healing: hemostasis, inflammation, repair, and encapsulation. Bacterial infection that can occur due to bacterial adhesion is a serious problem that affects implanted materials during healing or in the case of extended periods of time (Dankert et al., 1986, as cited in Shin & Schoenfisch, 2006; Sawan & Manivannan, 2000, as cited in Shin & Schoenfisch, 2006). In order to overcome this problem, strategies are being developed based on control release of an anticoagulant or antimicrobial agent from implanted material (Christiansen et al., 2011; Hendricks et al., 2000; Shin & Schoenfisch,

Drug delivery systems (DDS) are new types of drug carriers, designed to improve the pharmacological and therapeutic properties of administrated drugs (Allen & Cullis, 2004). Such systems are designed according to the type of administration. There are transdermal drug delivery systems (TDDS) used for external administration of drugs and liposomal and targeted drug delivery systems (LTDDS) for internal administration. TDDS present the advantages of avoiding hepatic first pass metabolism, of decreasing gastrointestinal effect and improving compliance, while LTDDS are used to reduce the dose and increase the activity of the drug at the precise site (Kshirsagar, 2000). DDS can be classified after their

The dose released can differ according to the type of the system used and can be: extended

Generally, DDS present zero-order kinetics and they should be inert and biocompatible with the environment. As compared to the conventional way of drug administration, DDS have the advantages of being more comfortable for the user, easily administrated, possess a high drug concentration, have good mechanical strength, safe and free from leaks. They also have biocompatible, non-toxic, nonmutagenic, non-carcinogenic, non-teratogenic and

the number of the cells increases.

2006; Williams, 2009).

release mechanism into (Lager & Peppas, 1981):

a. Reservoirs (membrane devices) b. Matrices (monolithic devices) 2. Chemically Controlled Systems c. Bioerodible systems d. Pendant Chain systems 3. Swelling Controlled Systems 4. Magnetically Controlled Systems

release, slow release and sustained release (Kshirsagar, 2000).

nonimmunogenic properties (Lager & Peppas, 1981).

1. Diffusion Controlled Systems

One of the mostly researched biomaterials used in various fields of medicine is natural polymer-collagen. Collagen is the most abundant protein in the body (skin, bones, teeth, tendons, cartilage, basement membrane, cornea, etc.). It can be used by different processing techniques in large various molecular structures (micro and nanostructures) as powder, injectable solutions, films, membrane and matrices (sponges).

The use of collagen as biomaterial, biocompatible and bioresorbable for the connective tissues prosthesis in which collagen is the basic protein is very well known.

To use collagen as scaffold in the recovery of bone tissue modifications in the structure and composition of matrix we need to obtain osteoinductive and osteoconductive effect. This can be achieved by the formation of biocomposites with SiO2, TiO2, clay, hydroxyapatite, etc (Christiansen et al., 2011; Vuluga et al., 2008a). Osteoinduction is the effect that appears in bone healing and involves the recruitment and stimulation of immature cells to develop into preosteoblast cells. Osteoconduction refers to bone growth on an implanted material, including bone conduction on its surface or down into pores, channels or pipes (Albrektsson & Johansson, 2001). Osteoconductive effect depends not only on biological factors, but also on the response to a foreign material. Bone conduction is not possible on certain materials, like some metals (Cu, Ag) (Albrektsson, 1995, as cited in Albrektsson & Johansson, 2001), while osteoinductive effect was observed on a large number of biomaterials (Barradas et al., 2011).

The layered silicate improves the thermal and mechanical properties of collagen and has a biostimulative effect on cellular metabolism. The structure of natural silicate would be like the structure of the living tissue because of the ionic exchange properties in aqueous medium just like the physiological phenomena in the human body where permanent information exchanges are present. Silicates surface can be modified with different bioactive substances and obtained nanohybrids can be dispersed in a collagen matrix (Potarniche et al., 2010; Potarniche et al. 2011). Depending on the concentration and the method of the silicate incorporation in the collagen protein, nanostructured systems with different morphologies, capable to control release of active principles (antibiotics, anti-inflammatory, enzymes, etc.) can be obtained. These types of nanocomposites can be used in regenerative medicine of bone tissue (Christiansen et al., 2011).

Polymer/inorganic nanocomposites are materials with unique properties which have found applications in large fields of activity: aircraft industry, automobile, packaging, construction, electronics, electrical engineering and last but not least in medicine and pharmaceutical industry. To obtain nanocomposites hybrids, the clay minerals (hydrated layered silicates) are preferred due to their high mechanical and chemical strength and low cost. The biomedical properties of clays are known since antiquity, these being in principal antiseptic, bactericides, scar action, antitoxic, without microbial germs. The special surface physical and chemical properties, such as high specific surface, layer charge, swelling capacity, make clay and clay minerals to be useful for human health in both external and "per os" (internal) applications. Clays can be used as active principles or excipients in pharmaceutical formulas, cosmetic medicine, in treating intestinal disorders and as drugs, being good adsorbents and mucostabilizers (Choy et al., 2007).

In medicine, synthetic polymers are also frequently used, not because of their biological activity, but because they are relatively easy to obtain, they are inert to body fluids, they are

Collagen - Modified Layered Silicate Biomaterials for Regenerative Medicine of Bone Tissue 131

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

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

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

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

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 &

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

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

microfibrils and fibrils. The collagen obtained is a gel, type I fibrillar collagen.

collagen biomaterials.

**2.1.3 Maleic copolymers** 

Chemistry, Romanian Academy, Iasi.

measurements in acetone at 30 °C.

Burger, 1970 as cited in Chitanu et al., 2006).

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

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

obtained by purification of Romanian sodium bentonite.

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., 2009).

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 for bone tissue regeneration.

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, antimicrobial antioxidants, etc.).

Obtaining of collagen/modified layered silicate nanocomposites implies:


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 regenerative medicine of bone tissue can be obtained.
