**2.Toxicological effect of the water-soluble monomers**

#### **2.1 Monomers**

Monomer is a molecule of any of a class of compounds, mostly organic, that can react with other molecules of the same or other compound to form very large molecules, or polymers. The essential feature of a monomer is polyfunctionality, the capacity to form chemical bonds to at least two other monomer molecules. Bifunctional monomers can form only linear, chainlike polymers, but monomers of higher functionality yield cross-linked, network polymeric products. Toxicological effects of the monomers are changing from very low (zero) to very high.

Some polymeric biomaterials such as hydrogels are produced by the effect of initiator such as chemical initiator, heat, light or high energy radiation from the water soluble-monomers.

#### **2.2 Cytotoxic effects**

Biomaterial suitable for a biomedical application must be biocompatible at least on its surface. In several previous studies, we investigated whether acrylamide, methacrylamide, N-isopropylacrylamide, acrylic acid, 2-hydroxyethyl methacrylate, 1-vinyl-2-pyrrolidone and ethylene glycol used in polimeric biomaterial production had cytotoxic effects (Unver Saraydin et al., 2011). The cytotoxicity of xenograft (one of the alternative graft materials) was also examined in vitro (Unver Saraydin et al., 2011).

The viability of cultured fibroblastic cell lines following all monomer applications except for the ethylene glycol group were found to be decreased in all time intervals (Figure 1, 2), and differences were statistically significant (p<0.05). In addition, the cell viability was significantly (p<0.05) lower in the acrylamid application group when compared to the control group. Acrylic acid demonstrated the maximum cytotoxic effect when compared to the methacrylamide and ethylene glycol groups. On the other hand, the ethylene glycol group showed no cytotoxicity for cells (Graphic 1).

In our study of the xenograft cytotoxic activities, the xenograft showed no cytotoxicity for the cells (Figure 3). There was no decolorization zone around the samples. Although the cells were directly in contact with the xenograft in the culture media, they did not show any signs of injury and preserved their morphological characteristics and wholeness like those seen in the controls.

In our previous other works, *in vitro* swelling and *in vivo* biocompatibility of radiation crosslinked acrylamide and its co-polymers such as acrylamide (AAm) and acrylamide/crotonic acid (AAm/CA), acrylamide/itaconic acid (AAm/IA), and

The radiation crosslinked AAm, AAm/CA, AAm/IA and (AAm/MA) co-polymers were

On the other hand, calcium phosphate ceramics and xenografts have been used in different fields of medicine and dentistry. We demonstrated the effects of calcium phosphate ceramics (Ceraform) and xenograft (Unilab Surgibone) in the field of experimentally created critical size parietal bone defects in rats. Although Ceraform was less resorptive and not osteoconductive properties, it could be considered as a biocompatible bone defect filling material having a limited application alternative in dentistry and medicine. However, xenograft seems biocompatible, osteoconductive, and could be used in a limited manner as a

Monomer is a molecule of any of a class of compounds, mostly organic, that can react with other molecules of the same or other compound to form very large molecules, or polymers. The essential feature of a monomer is polyfunctionality, the capacity to form chemical bonds to at least two other monomer molecules. Bifunctional monomers can form only linear, chainlike polymers, but monomers of higher functionality yield cross-linked, network polymeric products. Toxicological effects of the monomers are changing from very low

Some polymeric biomaterials such as hydrogels are produced by the effect of initiator such as chemical initiator, heat, light or high energy radiation from the water soluble-monomers.

Biomaterial suitable for a biomedical application must be biocompatible at least on its surface. In several previous studies, we investigated whether acrylamide, methacrylamide, N-isopropylacrylamide, acrylic acid, 2-hydroxyethyl methacrylate, 1-vinyl-2-pyrrolidone and ethylene glycol used in polimeric biomaterial production had cytotoxic effects (Unver Saraydin et al., 2011). The cytotoxicity of xenograft (one of the alternative graft materials)

The viability of cultured fibroblastic cell lines following all monomer applications except for the ethylene glycol group were found to be decreased in all time intervals (Figure 1, 2), and differences were statistically significant (p<0.05). In addition, the cell viability was significantly (p<0.05) lower in the acrylamid application group when compared to the control group. Acrylic acid demonstrated the maximum cytotoxic effect when compared to the methacrylamide and ethylene glycol groups. On the other hand, the ethylene glycol

In our study of the xenograft cytotoxic activities, the xenograft showed no cytotoxicity for the cells (Figure 3). There was no decolorization zone around the samples. Although the cells were directly in contact with the xenograft in the culture media, they did not show any signs of injury and preserved their morphological characteristics and wholeness like those

acrylamide/maleic acid (AAm/MA) hydrogels were investigated.

found to be well tolerated, non-toxic and highly biocompatible.

**2.Toxicological effect of the water-soluble monomers** 

filling material in osseous defects in clinical practice.

was also examined in vitro (Unver Saraydin et al., 2011).

group showed no cytotoxicity for cells (Graphic 1).

**2.1 Monomers** 

(zero) to very high.

**2.2 Cytotoxic effects** 

seen in the controls.

Fig. 1. Fibroblast viability %100 after 12 h incubation period with ethylene glycol

Fig. 2. Fibroblast viability % 0 after 12h incubation period with N-isopropyl acrylamide

Graphic 1. Shows the cell viability alterations between groups in the fibroblastic cell lines by the time.

Histopatological Effect Characteristics of Various

Fig. 4. Control group GFAP immunoreactivity. GFAP40X

Fig. 5. GFAP immunoreactivity 6 week after Acrylic acid exposure. GFAP 40X

Fig. 6. GFAP immunoreactivity 2 week after Acrylamide exposure. GFAP 40X

Biomaterials and Monomers Used in Polymeric Biomaterial Production 429

Fig. 3. There is no cytotoxicity for the cells.

#### **2.3 Neurotoxic effects**

Several studies revealed neurotoxic effects as well as ataxi and muscle weakness caused by biomaterials on humans and on laboratory animals. It has been suggested that they cause axonal degeneration in central and peripheric nervous system (Barber et al., 2001).

Astrocytes are the stellate glial cells in the central nervous system, which play a major role in supporting neurons, scar formation and development and maintenance of the blood-brain barrier. The physiological and metabolic properties of astrocytes indicate that those cells are involved in the regulation of water, ions, neurotransmitters, and pH of the neuronal milieu (Montgomery 1994). They are also implicated in protection against toxic insults such as excitotoxicity and oxidative stress (Lamigeon et al., 2001). Glial fibrillary acidic protein (GFAP) is an intermediate filament protein found predominantly in astrocytes (McLendon 1994). Therefore it is important to determine the glial fibrillary acidic protein (GFAP) immunoreactivity in astrocytes for the evaluation of biomateials.

In our study, immunolocalization of glial fibrillary acidic protein (GFAP) was determined, and it was evaluated by using semi-quantitative morphometrical techniques (Unver Saraydin et al., 2011). GFAP immunoreactivity was found to be very strong in the methacrylamide, N-isopropylacrilamid, ethylene glycol and N-vinyl pyrrolidine application groups whereas it was weak in acrylic acid, acrylamide and 2-hydroxyethyl metacrylad applied groups (Table 1, Figure 4-10). Changes in GFAP immunoreactivity could be due to following conditions; astrocyte dysfunction, astrocyte loss accompanied by astroglial cell proliferation, de-differentiation, and changes in functional state of neuronal cell types, thus altering the neuron-glial homeostasis. The over-expression of GFAP could probably indicate the protective strategy of these tissues.

Although the neurotoxicity of acrylamide and many monomers has been known since 1950s, its' mechanisms have remained obscure (Lee et al., 2005, Gold and Schaumburg, 2000). Acrylamide increases p53 protein (Okuno et al., 2006), recent studies indicate that it plays a role in apoptotic cell death in neurons (Morrison et al., 2003). Acrylamide can activate caspase- 3 and cause apoptosis in neuronal cells (Sumizawa and Igisu, 2007). The cellular process of apoptosis is an important component of tissue and organ development as well as the natural response to disease and injury (David et al., 2003). DNA fragmentation in neurons was characterized by double staining with terminal deoxynucleotidyl transferasemediated deoxyuridine triphosphate-biotin nick end labeling (TUNEL) (Bao and Liu, 2004). To our knowledge, however, it has not been determined whether acrylamide and other

Several studies revealed neurotoxic effects as well as ataxi and muscle weakness caused by biomaterials on humans and on laboratory animals. It has been suggested that they cause

Astrocytes are the stellate glial cells in the central nervous system, which play a major role in supporting neurons, scar formation and development and maintenance of the blood-brain barrier. The physiological and metabolic properties of astrocytes indicate that those cells are involved in the regulation of water, ions, neurotransmitters, and pH of the neuronal milieu (Montgomery 1994). They are also implicated in protection against toxic insults such as excitotoxicity and oxidative stress (Lamigeon et al., 2001). Glial fibrillary acidic protein (GFAP) is an intermediate filament protein found predominantly in astrocytes (McLendon 1994). Therefore it is important to determine the glial fibrillary acidic protein (GFAP)

In our study, immunolocalization of glial fibrillary acidic protein (GFAP) was determined, and it was evaluated by using semi-quantitative morphometrical techniques (Unver Saraydin et al., 2011). GFAP immunoreactivity was found to be very strong in the methacrylamide, N-isopropylacrilamid, ethylene glycol and N-vinyl pyrrolidine application groups whereas it was weak in acrylic acid, acrylamide and 2-hydroxyethyl metacrylad applied groups (Table 1, Figure 4-10). Changes in GFAP immunoreactivity could be due to following conditions; astrocyte dysfunction, astrocyte loss accompanied by astroglial cell proliferation, de-differentiation, and changes in functional state of neuronal cell types, thus altering the neuron-glial homeostasis. The over-expression of GFAP could probably indicate

Although the neurotoxicity of acrylamide and many monomers has been known since 1950s, its' mechanisms have remained obscure (Lee et al., 2005, Gold and Schaumburg, 2000). Acrylamide increases p53 protein (Okuno et al., 2006), recent studies indicate that it plays a role in apoptotic cell death in neurons (Morrison et al., 2003). Acrylamide can activate caspase- 3 and cause apoptosis in neuronal cells (Sumizawa and Igisu, 2007). The cellular process of apoptosis is an important component of tissue and organ development as well as the natural response to disease and injury (David et al., 2003). DNA fragmentation in neurons was characterized by double staining with terminal deoxynucleotidyl transferasemediated deoxyuridine triphosphate-biotin nick end labeling (TUNEL) (Bao and Liu, 2004). To our knowledge, however, it has not been determined whether acrylamide and other

axonal degeneration in central and peripheric nervous system (Barber et al., 2001).

immunoreactivity in astrocytes for the evaluation of biomateials.

Fig. 3. There is no cytotoxicity for the cells.

the protective strategy of these tissues.

**2.3 Neurotoxic effects** 

Fig. 4. Control group GFAP immunoreactivity. GFAP40X

Fig. 5. GFAP immunoreactivity 6 week after Acrylic acid exposure. GFAP 40X

Fig. 6. GFAP immunoreactivity 2 week after Acrylamide exposure. GFAP 40X

Histopatological Effect Characteristics of Various

Acrylamide ++ ++

cord (Unver Saraydin et al., 2011).

Biomaterials and Monomers Used in Polymeric Biomaterial Production 431

Fig. 10. GFAP immunoreactivity 4 week after N-vinyl pyrrolidone exposure. GFAP 40X

Methacrylamide +++ +++ ++ ++ + N-isopropylacrylamide +++ +++ +++ +++ Acrylic acid + ++ ++ ++ Control +++ +++ +++ +++ +++ Table 1. Demonstrates the semi-quantitative scoring findings of GFAP immunolocalization in rat medullaspinalis following 1, 2, 4, 6 and 12 weeks of particular monomer applications monomers cause apoptosis in neuronal cells. We therefore examined apoptosis by using terminal deoxynucleotydil transferase dUTP nick and labelling (TUNEL) method in spinal

While TUNEL positive cells has been detected rarely in the control and in the ethylen glycol application groups, numerous TUNEL positive cells were intensively observed in the spinal cord of the methacrylamide, acrylic acid, N-vinyl pyrrolidine, acrylamide, Nisopropylacrylamide and 2-hydroxyethyl metacrylate application groups (Figure 11-14).

Fig. 11. TUNEL-positive apoptotic cells in the control group. TUNEL 100X

Monomer 1st week 2nd week 4th week 6 th week 12 th week Ethylene glycol ++ ++ +++ ++ ++ N-vinyl pyrrolidone +++ +++ +++ +++ ++ 2-hydroxyethyl methacrylate + ++ +++ ++ ++

Fig. 7. GFAP immunoreactivity 6 week after 2-hydroxyethyl methacrylate exposure GFAP 40X

Fig. 8. GFAP immunoreactivity 4 week after methacrylamide exposure GFAP 40X

Fig. 9. GFAP immunoreactivity 6 week after N-isopropylacrylamide exposure GFAP 40X

Fig. 7. GFAP immunoreactivity 6 week after 2-hydroxyethyl methacrylate exposure GFAP 40X

Fig. 8. GFAP immunoreactivity 4 week after methacrylamide exposure GFAP 40X

Fig. 9. GFAP immunoreactivity 6 week after N-isopropylacrylamide exposure GFAP 40X

Fig. 10. GFAP immunoreactivity 4 week after N-vinyl pyrrolidone exposure. GFAP 40X


Table 1. Demonstrates the semi-quantitative scoring findings of GFAP immunolocalization in rat medullaspinalis following 1, 2, 4, 6 and 12 weeks of particular monomer applications

monomers cause apoptosis in neuronal cells. We therefore examined apoptosis by using terminal deoxynucleotydil transferase dUTP nick and labelling (TUNEL) method in spinal cord (Unver Saraydin et al., 2011).

While TUNEL positive cells has been detected rarely in the control and in the ethylen glycol application groups, numerous TUNEL positive cells were intensively observed in the spinal cord of the methacrylamide, acrylic acid, N-vinyl pyrrolidine, acrylamide, Nisopropylacrylamide and 2-hydroxyethyl metacrylate application groups (Figure 11-14).

Fig. 11. TUNEL-positive apoptotic cells in the control group. TUNEL 100X

Histopatological Effect Characteristics of Various

**3. Polymeric biomaterials** 

et.al. 2003).

Biomaterials and Monomers Used in Polymeric Biomaterial Production 433

Some polymeric biomaterials such as hydrogels are made of water-soluble molecules, connected usually by covalent bonds, forming a three-dimensional insoluble network. The space between chains is accessible for diffusion of solutes and this space is controllable by the level of cross-linked (connected) molecules. They usually show good biocompatibility in contact with blood, body fluids, and tissues. Therefore, they are very often used as biomaterials for medical purposes, for instance contact lenses, coating of catheters, etc. Biomaterials are defined as materials that can be interfaced with biological systems in order

The clinical application of a biomaterial should not cause any adverse reaction in the organism and should not endanger the life of the patient; any material to be used as part of a biomaterial device has to be biocompatible. The definition of biocompatibility includes that the material has to be nontoxic, non-allergenic, noncarcinogenic, and non-mutagenic, and that it does not influence the fertility of a given patient. Preliminary use of in vitro methods is encouraged as screening tests prior to animal testing. In order to reduce the number of animals used, these standards use a step-wise approach with review and analysis of test results at each stage. Appropriate in vitro investigations can be used for screening prospective biomaterials for estimations of toxic effect. Cytotoxicity in vitro assay is the first test to evaluate the biocompatibility of any material for use in biomedical devices (Rogero

Hydrogels can be synthesized by accomplishing crosslinking via -irradiation (Guven, O; et.al. 1999, Saraydn et.al. 1995, 2002, Karadağ et. al. 2004). However, little work is done on the biomedical applications of the hydrogels prepared by crosslinking of a homo- or copolymer in solution with -irradiation. It is well known that the presence of an initiator and a crosslinking agent affects the macromolecular structure and phase behavior of hydrophilic polymers in solution and contributes to inhomogeneity of the network structure. It is argued that more homogeneous network structures can be synthesized, if crosslinking is accomplished with -irradiation in the absence of an initiator and a crosslinking agent. The structural homogeneity of the network affects the swelling behavior and mechanical properties that improved the biological response of materials and subsequently the performance of many medical devices (Benson 2002). Thus, looking to the significant consequences of biocompatibility of biomaterials, we, in the present study, are reporting the results on the biocompatibility with the copolymeric hydrogels prepared with acrylamide (AAm) and crotonic acid (CA) or itaconic acid (IA) or maleic acid (MA) via radiation technique. The selection of AAm as a hydrophilic monomer for synthesizing hydrogel rests upon the fact that it has low cost, water soluble, neutral and biocompatible, and has been extensively employed in biotechnical and biomedical fields. On the other hand, CA monomer consists of single carboxyl group, while IA and MA monomers are consisting of double carboxyl groups. These carboxylic acids could provide the different functional characteristics to acrylamide-based hydrogels. So, these monomers were selected

for the preparation of the hydrogels and their biocompatibility studies.

In our previous other works, *in vitro* swelling and biocompatibility of blood *in vivo* biocompatibility of radiation crosslinked acrylamide co-polymers such as acrylamide (AAm), acrylamide/crotonic acid (AAm/CA), acrylamide/itaconic acid (AAm/IA) and

to evaluate, treat, augment, or replace any tissue, organ, or function of the body.

Fig. 12. TUNEL positive cells 6 week after 2-hydroxyethyl methacrylate. TUNEL 100X

Fig. 13. TUNEL positive cells 6 week after N-isopropylacrylamide. TUNEL 100X

Fig. 14. TUNEL positive cells 6 week after N-vinyl pyrrolidine. TUNEL 100X
