**3. Polymeric biomaterials**

432 Biomaterials – Physics and Chemistry

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

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

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 et.al. 2003).

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

Histopatological Effect Characteristics of Various

Normal

Biochemical parameters of human serum / Unit

Biomaterials and Monomers Used in Polymeric Biomaterial Production 435

Glucose/mg dl-1 70-110 87.0±8.2 91.0± 6.1 88.1±3.99 88.8±6.0 88.4±5.30 Triglyceride/mg dl-1 40-160 127.3±24.6 127.0±25.8 130.6±19.9 127.2±25.1 125.6±20.7 Cholesterol/mg dl-1 125-350 158.6±10.9 160.6±14.3 159.8±11.3 157.8±10.8 160.6±14.3 BUN/mg dl-1 8-25 14.8±1.27 15.2±4.56 14.6±3.73 15.2±4.10 15.6±3.84 Creatinin/mg dl-1 0.8-1.6 0.98±0.14 1.06±0.17 1.02±0.14 0.98±0.18 1.00±0.18 Total protein/g dl-1 6.0-8.4 6.52±0.15 6.72±0.15 6.70±0.13 6.60±0.22 6.48±0.30 Albumin/mg dl-1 3.5-5.6 4.02±0.15 3.88±0.15 3.98±0.18 3.96±0.20 3.94±0.10 Alkaline phosphatase/U 35-125 53.6± 13.1 54.5±12.3 54.0±14.9 52.6±12.6 52.6± 10.3 Alanine transaminase/U 7-56 14.6±2.12 16.0±2.63 15.7±3.23 15.9±2.47 16.0±2.63 Aspartate transaminase/U 5-40 16.2±5.33 15.2±3.19 16.5±3.03 17.2±5.16 15.2±3.19 Direct bilirubin/mg dl-1 0.0-0.3 0.12±0.04 0.12±0.04 0.11±0.03 0.11±0.03 0.12±0.04 Indirect bilirubin/mg dl-1 0.1-1.1 0.45±0.05 0.35±0.09 0.35±0.09 0.45±0.05 0.40±0.07 Chlorine/meq dl-1 95-107 98.5±2.17 98.8±2.3 98.6±2.12 97.8±1.75 98.2±2.10 Sodium/meq dl-1 137-146 142.7±1.4 142.8± 0.9 143.0±1.6 142.7±1.4 142.0±1.6 Potassium/meq dl-1 3.5-5.5 4.80±0.28 4.68±0.36 4.94±0.39 4.87±0.35 4.70±0.35 Calcium/mg dl-1 8.5-10.8 9.40±0.39 9.47±0.28 9.42±0.28 9.63±0.42 9.47±0.28 Phosphorus/mg dl-1 2.5-4.5 3.60±0.41 3.60±0.32 3.68±0.42 3.60±0.36 3.56±0.38

Table 3. Means and standard deviations of biochemical parameters of human sera

were observed between tissues and capsule in the some samples (Figure 15, 16).

Fig. 15. After one week, the implan-tation site of AAm hydrogel, H-E, 20X

skin and the tissues of straight muscle in the close to implant sites.

After 6–10 weeks, the adverse tissue reaction, giant cells and necrosis of cells, inflammatory reaction such as deposition of foamed macrophage were not observed in the implant site, however, it is observed to increase in the collagen fibrils due to proliferation and activation of fibroblasts (Fig. 17). No chronic and acute inflammation, adverse tissue reaction were observed in the all test groups. It is no determination related to the loss of activation and liveliness of cells in the capsule cells and in distant sites. No pathology were observed in the

these fibrous capsules consisting of fibroblasts, and a grouped mast cells and lymphocyte

values Control AAm AAm/CA AAm/MA AAm/IA

acrylamide/maleic acid (AAm/MA) hydrogels were investigated (Saraydin et al., 1995, Karadağ et. al. 1996, Saraydin et al., 2001, 2004).

#### **3.1 In vitro swelling of the hydrogels in the simulated body fluids**

In this stage of the study, the swelling of the hydrogels in the simulated physiological body fluids was investigated (Saraydin et al., 1995, Karadağ et. al. 1996).

The phosphate buffer at pH 7.4 (pH of cell fluid, plasma, edema fluid, synovial fluid, cerebrospinal fluid, aqueous humour, tears, gastric mucus, and jejunal fluid), glycine-HCl buffer at pH 1.1 (pH of gastric juice), human sera, physiological saline and distilled water intake of initially dry hydrogels were followed for a long time until equilibrium (Saraydin et al., 2001, 2002).

The fluid absorbed by the gel network is quantitatively represented by the EFC (equilibrium body fluids content), where: EFC% = [mass of fluid in the gel/mass of hydrogel] x 100. EFCs of the hydrogels for all physiologically fluids were calculated. The values of EFC% of the hydrogels are tabulated in Table 2.


Table 2. EFC values of the hydrogels

All EFC values of the hydrogels were greater than the percent water content values of the body about 60%. Thus, the AAm and AAm/CA, AAm/MA and AAm/IA hydrogels were exhibit similarity of the fluid contents with those of living tissues.

#### **3.2 In vitro blood biocompatibility**

In the second stage of this study, the biocompatibility of the hydrogels was investigated against some biochemical parameters of human sera at 25 OC.

The mean and standard deviation values of control and test groups for biochemical parameters of human sera are listed in Table 3*.* 

Table 3 shows that the values of means of control and test groups are in the range of normal values and there is no significant difference in values before and after contacting these sera with the hydrogels. On the other hand, Student's t-test is applied to control and test groups. No significant difference in values of biochemical parameters was found.

#### **3.3 In vivo tissue biocompatibility**

In this part, hydrogels based on copolymer of AAm, AAm/MA, AAm/CA and AAm/IA with capacity of absorbing a high water content in biocompatibility with subcutaneous tissues of rats were examined. After one week implantation, no pathology such as necrosis, tumorigenesis or infection were observed in the excised tissue surrounding the hydrogels and in skin, superficial fascia and muscle tissues in distant sites. After 2–4 weeks, thin fibrous capsules were thickened. A few macrophage and lymphocyte were observed in

acrylamide/maleic acid (AAm/MA) hydrogels were investigated (Saraydin et al., 1995,

In this stage of the study, the swelling of the hydrogels in the simulated physiological body

The phosphate buffer at pH 7.4 (pH of cell fluid, plasma, edema fluid, synovial fluid, cerebrospinal fluid, aqueous humour, tears, gastric mucus, and jejunal fluid), glycine-HCl buffer at pH 1.1 (pH of gastric juice), human sera, physiological saline and distilled water intake of initially dry hydrogels were followed for a long time until equilibrium (Saraydin et

The fluid absorbed by the gel network is quantitatively represented by the EFC (equilibrium body fluids content), where: EFC% = [mass of fluid in the gel/mass of hydrogel] x 100. EFCs of the hydrogels for all physiologically fluids were calculated. The values of EFC% of the

Simulated body fluid AAm AAm /CA AAm /MA AAm /IA Distilled Water 86.3 93.9 94,7 92.0 Isoosmotic phosphate buffer 87.5 93.8 89,7 92.2 Gastric fluid 87.7 93.6 92,4 88.7 physiological saline 87.8 92.9 89,7 88.7 Human Sera 88.6 92.5 89.8 86.4 In rat 89.0 93.1 91.9 91.7

All EFC values of the hydrogels were greater than the percent water content values of the body about 60%. Thus, the AAm and AAm/CA, AAm/MA and AAm/IA hydrogels were

In the second stage of this study, the biocompatibility of the hydrogels was investigated

The mean and standard deviation values of control and test groups for biochemical

Table 3 shows that the values of means of control and test groups are in the range of normal values and there is no significant difference in values before and after contacting these sera with the hydrogels. On the other hand, Student's t-test is applied to control and test groups.

In this part, hydrogels based on copolymer of AAm, AAm/MA, AAm/CA and AAm/IA with capacity of absorbing a high water content in biocompatibility with subcutaneous tissues of rats were examined. After one week implantation, no pathology such as necrosis, tumorigenesis or infection were observed in the excised tissue surrounding the hydrogels and in skin, superficial fascia and muscle tissues in distant sites. After 2–4 weeks, thin fibrous capsules were thickened. A few macrophage and lymphocyte were observed in

Karadağ et. al. 1996, Saraydin et al., 2001, 2004).

al., 2001, 2002).

hydrogels are tabulated in Table 2.

Table 2. EFC values of the hydrogels

**3.2 In vitro blood biocompatibility** 

**3.3 In vivo tissue biocompatibility** 

parameters of human sera are listed in Table 3*.* 

**3.1 In vitro swelling of the hydrogels in the simulated body fluids** 

fluids was investigated (Saraydin et al., 1995, Karadağ et. al. 1996).

exhibit similarity of the fluid contents with those of living tissues.

against some biochemical parameters of human sera at 25 OC.

No significant difference in values of biochemical parameters was found.


Table 3. Means and standard deviations of biochemical parameters of human sera

these fibrous capsules consisting of fibroblasts, and a grouped mast cells and lymphocyte were observed between tissues and capsule in the some samples (Figure 15, 16).

Fig. 15. After one week, the implan-tation site of AAm hydrogel, H-E, 20X

After 6–10 weeks, the adverse tissue reaction, giant cells and necrosis of cells, inflammatory reaction such as deposition of foamed macrophage were not observed in the implant site, however, it is observed to increase in the collagen fibrils due to proliferation and activation of fibroblasts (Fig. 17). No chronic and acute inflammation, adverse tissue reaction were observed in the all test groups. It is no determination related to the loss of activation and liveliness of cells in the capsule cells and in distant sites. No pathology were observed in the skin and the tissues of straight muscle in the close to implant sites.

Histopatological Effect Characteristics of Various

methacrylate) hydrogels for the biomedical uses.

**4. Bioactive ceramic biomaterials** 

bioglass.

Graphic 2*.* The curves of thickness of fibrous capsule—implantation time.

mandibular bone defects in rats (Develioglu et al., 2006, 2007, 2009, 2010).

Bioactive refers to a material, which upon being placed within the human body interacts with the surrounding bone and in some cases, even soft tissue. This occurs through a time – dependent kinetic modification of the surface, triggered by their implantation within the living bone. An ion – exchange reaction between the bioactive implant and surrounding body fluids – results in the formation of a biologically active carbonate apatite (CHAp) layer on the implant that is chemically and crystallographically equivalent to the mineral phase in bone. Prime examples of these materials are synthetic hydroxyapatite, glass ceramic and

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 and

Many researches are currently conducted to find out the ideal material to support bone repair or regeneration. The limitations of autogenous grafts and allogeneic bankbone have led to a search for synthetic alloplast alternatives. Calcium phosphate ceramics have been

Biomaterials and Monomers Used in Polymeric Biomaterial Production 437

found. These thickness of fibrous capsule indicated well within the critical tissue tolerance range. It was given by the some reporters that the threshold capsule thickness should not exceed 200–250 m for an implanted biomaterial (Jeyanthi and Rao, 1990). Our results clearly indicated that the capsule thickness of the excised tissue were well within these stipulated threshold limits. On the basis of the findings we can conclude that the biological response against the tested hydrogels was very similar to the biocompatibility of very low swollen of poly(2-hydroxyethyl methacrylate) hydrogel, which considered as a biologically inert polymer (Smetana et al., 1990). However, it is important that the swelling of acrylamide based hydrogels are very high than the swelling of poly(2-hydroxyethyl

Fig. 16. After 4 week, the implantation site of AAm hydrogel, H-E, 20X

Fig. 17. 10 week postimplantation of AAm/CA hydrogel. H-E, 20X

The thickness of the fibrous capsules were measured in the optical microscope using a micrometer scale. The means of five measurements for each the sample and each time point were calculated. The thickness of fibrous capsules are gradually increased to 6 weeks, and then these values are becomed a constant value. The thickness of fibrous capsule occurred due to AAm/CA, AAm/MA and AAm/IA hydrogels implant are high from the values of AAm and hydrogels. The carboxyl groups on the chemical structure and ionogenic character of AAm/CA, AAm/MA and AAm/IA hydrogels can be caused to the high thickness of the fibrous capsule (Smetana et al., 1990). The thickness of the fibrous capsules were measured in the optical microscope using a micrometer scale. The means of five measurements for each the sample and each time point were calculated and shown in Graphic 2. The thickness of fibrous capsules are gradually increased to 6 weeks, and then these values are becomed a constant value. The thickness of fibrous capsule occurred due to AAm/CA, AAm/MA and AAm/IA hydrogels implant are high from the values of AAm and hydrogels. The carboxyl groups on the chemical structure and ionogenic character of AAm/CA, AAm/MA and AAm/IA hydrogels can be caused to the high thickness of the fibrous capsule (Smetana et al., 1990). On the other hand, Student's *t* test was applied to the all constant values of thickness of fibrous capsules of the hydrogels, and no significant differences (*p >*0*:*05) was

Fig. 16. After 4 week, the implantation site of AAm hydrogel, H-E, 20X

Fig. 17. 10 week postimplantation of AAm/CA hydrogel. H-E, 20X

The thickness of the fibrous capsules were measured in the optical microscope using a micrometer scale. The means of five measurements for each the sample and each time point were calculated. The thickness of fibrous capsules are gradually increased to 6 weeks, and then these values are becomed a constant value. The thickness of fibrous capsule occurred due to AAm/CA, AAm/MA and AAm/IA hydrogels implant are high from the values of AAm and hydrogels. The carboxyl groups on the chemical structure and ionogenic character of AAm/CA, AAm/MA and AAm/IA hydrogels can be caused to the high thickness of the fibrous capsule (Smetana et al., 1990). The thickness of the fibrous capsules were measured in the optical microscope using a micrometer scale. The means of five measurements for each the sample and each time point were calculated and shown in Graphic 2. The thickness of fibrous capsules are gradually increased to 6 weeks, and then these values are becomed a constant value. The thickness of fibrous capsule occurred due to AAm/CA, AAm/MA and AAm/IA hydrogels implant are high from the values of AAm and hydrogels. The carboxyl groups on the chemical structure and ionogenic character of AAm/CA, AAm/MA and AAm/IA hydrogels can be caused to the high thickness of the fibrous capsule (Smetana et al., 1990). On the other hand, Student's *t* test was applied to the all constant values of thickness of fibrous capsules of the hydrogels, and no significant differences (*p >*0*:*05) was found. These thickness of fibrous capsule indicated well within the critical tissue tolerance range. It was given by the some reporters that the threshold capsule thickness should not exceed 200–250 m for an implanted biomaterial (Jeyanthi and Rao, 1990). Our results clearly indicated that the capsule thickness of the excised tissue were well within these stipulated threshold limits. On the basis of the findings we can conclude that the biological response against the tested hydrogels was very similar to the biocompatibility of very low swollen of poly(2-hydroxyethyl methacrylate) hydrogel, which considered as a biologically inert polymer (Smetana et al., 1990). However, it is important that the swelling of acrylamide based hydrogels are very high than the swelling of poly(2-hydroxyethyl methacrylate) hydrogels for the biomedical uses.

Graphic 2*.* The curves of thickness of fibrous capsule—implantation time.
