**4.2. Biocompatibility**

One of the most important factors that distinguish biomaterials is its ability to exist into or in contact with tissues of the human body without inducing any collateral effect, where both biomaterials and tissues coexist, and the biocompatibility may be compromised.

Biocompatibility refers to the ability of a material to perform with an appropriate host response in a specific situation [76].

The biocompatibility of a material depends on the type of material, where it is placed, and the function it is expected to perform. Therefore, a biocompatible material elicits an acceptable tissue response when tested or used in a specific tissue under certain conditions, including the health status of the patient [77]. It is important to understand the paradigms of biocompatibility by the determination of which chemical, biochemical, physiological, physical, or other mechanisms, under specific conditions, associated with contact between biomaterials and cells or tissues of the body. The interactions of materials that are in direct contact with the human body depends on the characteristics of the host such as age, sex, general health and current disease, physical mobility, lifestyle features, and pharmacological status [78]. Thus, the major features influenced in the host and generic host response (implanted or in contact with tissues) of biomaterials are enlisted in **Table 5**.

On the other hand, PMMA-based acrylic resin has been broadly used as a dental material, especially in denture base processing due to its favorable working characteristics, processing ease, accurate fit, stability in the oral environment, and superior aesthetics with inexpensive equipment. Despite these excellent properties, there is a need for improvement in the biological aspects of biocompatibility. This section is oriented to summarize the different methods of PMMA biocompatibility alone and enriched or modified with different biomaterials in contact with cells and implantation in animal bodies highlighting the type of cells or animal test, period of incubation or implantation, method for analysis, and results. The incorporated studies are recent publications indexed at MEDLINE/PUBMED based on a systematic review.

#### *4.2.1. Test methods*

Testing for cytocompatibility depends on the site of use and the duration of exposure. Biomaterials or other associated products do not have to exhibit the same compatibility as materials that are placed permanently into the tooth structure, used as implants into bone or soft tissues, or used in dentures and dental or orthodontic appliances. All the tests are usually conducted sequentially, with shorter term, *in vitro*, or less expensive screening testing, and involve the use of animals. If a material is not showing biocompatibility based on initial studies, it may be better to eliminate it from consideration for further testing for certain applications [79]. The biocompatibility concerns and the testing methods have been discussed for over 40 years. However, new issues and new testing possibilities must be considered for innovating dental materials and evaluate the response of cells to medical materials at the cellular


**Table 5.** Biomaterial variables that could influence the host response [80].

and subcellular levels such as cell proliferation or death in contact with materials [80]. These protocols and methods of interpretation may be used to enhance the information given in the National and International standards.

#### *4.2.1.1. Cell culture testing*

and mechanical properties as well [24]. In both the cases, specimens exhibited good mechanical and physical properties and were not non-cytotoxic showing similar appearance to com-

One of the most important factors that distinguish biomaterials is its ability to exist into or in contact with tissues of the human body without inducing any collateral effect, where both

Biocompatibility refers to the ability of a material to perform with an appropriate host response

The biocompatibility of a material depends on the type of material, where it is placed, and the function it is expected to perform. Therefore, a biocompatible material elicits an acceptable tissue response when tested or used in a specific tissue under certain conditions, including the health status of the patient [77]. It is important to understand the paradigms of biocompatibility by the determination of which chemical, biochemical, physiological, physical, or other mechanisms, under specific conditions, associated with contact between biomaterials and cells or tissues of the body. The interactions of materials that are in direct contact with the human body depends on the characteristics of the host such as age, sex, general health and current disease, physical mobility, lifestyle features, and pharmacological status [78]. Thus, the major features influenced in the host and generic host response (implanted or in contact

On the other hand, PMMA-based acrylic resin has been broadly used as a dental material, especially in denture base processing due to its favorable working characteristics, processing ease, accurate fit, stability in the oral environment, and superior aesthetics with inexpensive equipment. Despite these excellent properties, there is a need for improvement in the biological aspects of biocompatibility. This section is oriented to summarize the different methods of PMMA biocompatibility alone and enriched or modified with different biomaterials in contact with cells and implantation in animal bodies highlighting the type of cells or animal test, period of incubation or implantation, method for analysis, and results. The incorporated studies are recent publications indexed at MEDLINE/PUBMED based on a systematic review.

Testing for cytocompatibility depends on the site of use and the duration of exposure. Biomaterials or other associated products do not have to exhibit the same compatibility as materials that are placed permanently into the tooth structure, used as implants into bone or soft tissues, or used in dentures and dental or orthodontic appliances. All the tests are usually conducted sequentially, with shorter term, *in vitro*, or less expensive screening testing, and involve the use of animals. If a material is not showing biocompatibility based on initial studies, it may be better to eliminate it from consideration for further testing for certain applications [79]. The biocompatibility concerns and the testing methods have been discussed for over 40 years. However, new issues and new testing possibilities must be considered for innovating dental materials and evaluate the response of cells to medical materials at the cellular

biomaterials and tissues coexist, and the biocompatibility may be compromised.

mercial acrylic resins.

54 Acrylic Polymers in Healthcare

**4.2. Biocompatibility**

in a specific situation [76].

*4.2.1. Test methods*

with tissues) of biomaterials are enlisted in **Table 5**.

The most common and initial evaluation of a new material is by placing the material or an extract of the material into a suitable laboratory cell culture and by observing any changes in the cells over a period of hours to a few days [81]. These tests are performed on primary cell cultures or established cell lines (commercially available), which allows comparison of testing performed for different materials using nearly identical cloned cells. The use of PMMA acrylic base denture has been widely investigated in culture cells alone and enriched with different materials. The enlisted publications in **Table 6** was searched at MEDLINE/PUBMED with the following keywords: "Cytotoxicity AND acrylic resins," "cytotoxicity AND denture



**Author** Herman et al. [82]

**PMMA modification**

PMMA, monomer modified with

DABCO (DC16, DC16F, DC18, C6DC16)

and conjugated monomers (DC11MAF

and C2DC11MAF) at 1, 2, or 3%

Song et al. [83]

Liu et al. [84] da Silva et al. [85]

N1 acrylic (MMA polymer, dibutyl phthalate

Human conjunctiva

MTT

72 h

Non-cytotoxic based on cell

proliferation. Resin with

pigment showed significant

increase of IL6

ELISA

RT-PCR

cell line (CCL-20.2).

), ethyl acrylate pigments), Poli-Côr (color

R2, MMA polymer, dibutyl phthalate,

ethyl acrylate, around 1.5% of various

organic and inorganic pigments), Clássico

(MMA monomer, topanol)

PMMA-based bone cement-Osteopal V

Human osteoblast-like

AlamarBlue assay

24 h

*In vitro* cytotoxicity appeared

somewhat affected by the

castor oil and linoleic acid

additions

Fluorescence

Saos-2 cells (HPACC)

modified with castor oil and linoleic acid

Carlsson et al. [86]

Jiao et al. [87]

PMMA enriched with 15% of N-acetyl

Human dental pulp

Extracts preparation

3, 7 days

The addition of NAC

remarkably improved

biocompatibility of PMMA

resin

and MTT

cells

cysteine (NAC)

PMMA-PEI (polyethyleneimine)

Kupffer cells (KCs)

Cell Counting Kit-8

6, 12, 24, 48 h

primary culture

assay

Fluorescence

Western blot

nanoparticles

PMMA with chitosan (0.50, 1, 2, 3 mg/ml)

(L929)

Mouse fibroblast cells

MTT

0, 24, 48, 72 h

**Culture cells** Periodontal ligament

BioTek Synergy2

24 h

fluorescent

cells (PDL) and

gingival fibroblast

(HGF)

**Assays**

**Culture time**

**Results**

DABCO components

exhibit intermediate to high

cytotoxicity and DC11MAF

56 Acrylic Polymers in Healthcare

exhibited the lowest toxicity

against PDL and GF

No significant difference in

cell proliferation between

conventional resin and

the chitosan quaternary

ammonium salt modified

Exhibit survival fraction

higher than 90%. These results

suggested that the PEI-PMMA/miRNA-complexed NPs had low cytotoxicity to

KCs



**Author** Son et al. [94]

**PMMA modification**

Scaffolds were fabricated by

electrospinning using polycaprolactone

(PCL) blended with PMMA; extracts

solutions

Jiang et al. [95]

Neves et al. [96]

Tencomnao et al. [97]

Acosta-Torres et al.

PMMA enriched with Silver

NIH-3T3 mouse

MTT

24, 72 h

BrdU assay

embryonic cells

Mouse fibroblast cells

H-thymidine

24 h

Trusoft, lucitone 550 showed

slightly cytotoxic effect

Dentuflux showed moderate

cytotoxic effect when

materials were stored in water

non-cytotoxic effect

Thermal treatment did not

reduce the cytotoxicity effect

of the acrylic-based soft lines

incorporation assay

(L929)

nanoparticles (AgNPs)

Lucitone 550-HR

Soft-Liners:

Ufi-Gel P-Silicon

Dentuflux-AR

Trusoft-AR

Dentusoft-Tissue conditioner

Water storage time after polymerization

[98]

Tay et al. [99]

PMMA core/polyethyleneimine (PEI)

Human neuroblastoma

MTT

24 h

The viability of LAN-5 cells

after transfection was in the

range of 80–100%

Non-cytotoxic material

(LAN-5)

shell magnetic nanoparticles

Ethanol treatment postpolymerization

Human adult dermal

MTT

24 h

LDH assay

fibroblast cells

of PMMA, extracts test from heath

treatment and conventional

PMMA particles

Bone marrow stromal

MTT

1, 3, 5, 7 days

PMMA did not stimulate cell

proliferation effect even at

low doses (0.63mg/ml) and

the particles appeared to

exhibit certain cytotoxic effect

at high concentration

(3 mg/ml)

Specimens showed significant

reduction on cytotoxicity

compared to immersion in

hot water

CytoTox 96

ELISA

RT-PCR.

cells

**Culture cells** Human osteosarcoma

MTT

Western blot

cells (MG63)

**Assays**

**Culture time**

1, 5, 7 days

PCL/PMMA blends are

suitable for osteoblast cell

proliferation

58 Acrylic Polymers in Healthcare

**Results**

**Table 6.** Cytotoxicity test of PMMA alone or modified. base resins," and "cytotoxicity AND oral prosthesis." Inclusion criteria were: *in vitro* studies published from 2012 to 2017, free full text, and published in English evaluating the PMMA and its components, considering cytotoxicity activity, type of material tested, kinds of cells used, period of incubation, assay executed, and results of the biocompatibility. Two reviewers read the selected studies, and their information was analyzed and discussed. **Figure 5** shows the flow chart of search strategy and the total number of studies included.

#### *4.2.1.2. Mutagenicity testing*

A concern for any material used in medicine is that long-term exposure to a material can lead to neoplastic changes in cells adjacent to it. Most materials are known to be acceptable based on a history of use, but changes in formulations and innovation of new materials are necessary to re-execute testing. Small animal *in vivo* mutagenicity studies allow screening of materials in development and reduce the use of research animals. Mutagenicity studies (also called genotoxicity studies) involve looking for changes in cells and cellular DNA in the forward or reverse directions. In forward mutation studies, normal cells are exposed to the test material and the resultant cells or animal tissues are evaluated for signs of mutation. Just a few studies have been tested for genotoxicity between PMMA and culture or small number of animal evaluations has been conducted. **Table 7** summarizes the investigations performed between mutagenicity and PMMA. The keywords used for search strategy at MEDLINE/PUBMED were as follows: "Genotoxicity AND acrylic resin," "genotoxicity AND polymethylmethacrylate resin,"

**Figure 5.** Search strategy flow chart. Source: Direct.

New Trends for the Processing of Poly(Methyl Methacrylate) Biomaterial for Dental Prosthodontics http://dx.doi.org/10.5772/intechopen.69066 61


**Table 7.** Mutagenicity test evaluation of PMMA alone and modified with different materials.

"genotoxicity AND denture base resin," "mutagenicity AND acrylic resin," "mutagenicity AND denture base resin," "mutagenicity AND oral prosthesis," and "mutagenicity AND polymethylmethacrylate resin." The inclusion criteria were: publications from 2012 to 2017, free full text. Only a few studies are reported in literature, a further MeSH term search was executed "("Mutagenicity Tests"[Mesh]) AND "Acrylic Resins"[Mesh], ("Mutagenicity Tests"[Mesh]) AND "Denture Bases"[Mesh], and ("Mutagenicity Tests"[Mesh]) AND "Polymethyl Methacrylate"[Mesh]. **Figure 1** shows the flow chart of search strategy and the total number of studies included.

#### *4.2.1.3. Short- or long-term injection or implantation studies*

base resins," and "cytotoxicity AND oral prosthesis." Inclusion criteria were: *in vitro* studies published from 2012 to 2017, free full text, and published in English evaluating the PMMA and its components, considering cytotoxicity activity, type of material tested, kinds of cells used, period of incubation, assay executed, and results of the biocompatibility. Two reviewers read the selected studies, and their information was analyzed and discussed. **Figure 5** shows

A concern for any material used in medicine is that long-term exposure to a material can lead to neoplastic changes in cells adjacent to it. Most materials are known to be acceptable based on a history of use, but changes in formulations and innovation of new materials are necessary to re-execute testing. Small animal *in vivo* mutagenicity studies allow screening of materials in development and reduce the use of research animals. Mutagenicity studies (also called genotoxicity studies) involve looking for changes in cells and cellular DNA in the forward or reverse directions. In forward mutation studies, normal cells are exposed to the test material and the resultant cells or animal tissues are evaluated for signs of mutation. Just a few studies have been tested for genotoxicity between PMMA and culture or small number of animal evaluations has been conducted. **Table 7** summarizes the investigations performed between mutagenicity and PMMA. The keywords used for search strategy at MEDLINE/PUBMED were as follows: "Genotoxicity AND acrylic resin," "genotoxicity AND polymethylmethacrylate resin,"

the flow chart of search strategy and the total number of studies included.

*4.2.1.2. Mutagenicity testing*

60 Acrylic Polymers in Healthcare

**Figure 5.** Search strategy flow chart. Source: Direct.

Several different tests may be conducted to provide information on the effects of relatively short-term exposure to materials or their extracts. These tests include systemic injection, intracutaneous injection for irritation, and short- and long-term implant studies ranging from 24 hours to as long as 90 days or years [80]. Certain materials have the potential to cause local inflammation of tissues. A special case of long-term implantation studies involves the lifetime bioassay performed for investigation of carcinogenicity. These studies are usually performed in several hundred rats and mice to look for differences in tumor formation as a result of exposure to the test material [79]. These studies allow screening out of candidate materials that may not be suitable for further testing. **Table 8** shows the results of search strategy at MEDLINE/PUBMED. The search strategy was previously described in cells tested with the incisive criteria of animal test. **Figure 5** shows the flow chart of search strategy and the total number of studies included.


**Table 8.** Short- or long-term exposure test of PMMA alone or modified.

### *4.2.2. Acrylic resin cytotoxicity*

**Author** Carlsson et al. [86]

**Type of material**

PMMA-based bone cement-Osteopal V modified with castor

oil and linoleic acid

Tsuji et al. [106]

Liu et al. [84]

Yu [90]

PMMA enriched with calcium

SD rats bone defect

4 weeks, 15 weeks

X-ray

Histological observation

phosphate cement (CPC) at 3:1,

2:1, 1:1, 1:2, 1:5, 1:10, 1:15, and

1:20

Son et al. [94]

Scaffolds were fabricated

Sprague Dawley rats;

1 and 2 months

Micro CT

Histological observation

skull defects and PCL/

PMMA implantation

by electrospinning using

polycaprolactone (PCL) blended

with poly(methylmethacrylate)

(PMMA)

**Table 8.**

Short- or long-term exposure test of PMMA alone or modified.

PMMA-PEI nanoparticles

C57/BL6 mice

6 hours

Western blot

NF-κB P65 protein levels

in liver tissue

Coating with titanium dioxide

Hamster oral mucosa

Irritation test: 24 h

Histological analysis

Skin sensitization: 2

irritation test, a guinea

pig skin sensitization

days

Intracutaneous test: 24,

48, and 72 h

test and a rabbit

intracutaneous test

(TiO2) nanoparticles

**Animal model**

Male Sprague-Dawley

1, 4, 12 weeks

rats

**Implantation time**

**Analysis** Flow cytometry

Histological analysis

**Results**

No differences could be

found in the

*in vivo* response to these

62 Acrylic Polymers in Healthcare

PMMA-based cements

The PMMA coated with

TiO2 NPs does not cause

irritation or sensitization

of the oral mucosa, skin,

or intracutaneous tissue

and is therefore good

PMMA-PEI NPs

could induce targeted

transfection (34.7%)

Except for the PMMA

group significant

degradations appeared

in both the CPC/PMMA

group (50%; 1:1) and

CPC group. Enhanced

the bone cell growth

Bone formation was

observed on the 7/3

PCL/PMMA scaffold

within 2 months

Different methods are used for cytotoxicity, mutagenicity, and short- or long-term implantation analysis in the literature. Among them, the most common is the MTT test [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium] and a histological evaluation. In the case of MTT, the method quantifies the mitochondrial succinate dehydrogenase enzyme activity and measures the conversion of water-soluble tetrazolium salt in insoluble blue formazan by spectrophotometry. This test is an excellent marker of cell survival because it evaluates cellular respiratory activity [100, 107].

The cytotoxicity of PMMA is correlated with the polymerization methods, temperature, the cycle of polymerization, and acrylic resin storage time can influence the monomer quantity and the material cytotoxicity [95, 100]. Based on the polymerization method, acrylic resin can be classified as heat-polymerized, microwave-polymerized, light-polymerized, and autopolymerized MMA. The latter being the most commonly used in dental practice [15]. The autopolymerized resin exhibited higher cytotoxicity level than heat-polymerized resin after 1 and 24 h of incubation [100]. The experiment performed of PMMA alone in contact with HGF showed similar results of dose-dependent cytotoxicity. It is important to use polished acrylic resins for clinical applications. The unpolished acrylic resin showed cell growth reduction, and an increase in pro-inflammatory cytokines were caused by the tested material [93, 102].

Postpolymerization heat treatments, such as water bath or microwave irradiation, have been suggested in order to reduce the quantity of autopolymerized acrylic resin residual monomers. The PMMA that was immersed into water showed a reduction in MMA monomer elucidation [88, 96].

Several substances such as chitosan [83], PEI (polyethyleneimine) nanoparticles [84], 15% of N-acetyl cysteine (NAC) [87], calcium phosphate cement (CPC) [90], acrylic resin of different colors [91], (bone cement) enriched with microencapsulated 2-octyl cyanoacrylate (OCA), extracts solutions [92], scaffolds fabricated by electrospinning using polycaprolactone (PCL) [94], core/polyethyleneimine (PEI) shell magnetic nanoparticles [97], silver nanoparticles (AgNPs) [98], and Paladon 65-HR precoated with biosurfactant were also evaluated for cytotoxicity. The authors observed reduction in cytotoxicity and increase in biocompatibility from non-cytotoxic (cell viability higher than 75%) to slightly cytotoxic (cell viability ranging from 50 to 75%)

On the other hand, DABCO (DC16, DC16F, DC18, C6DC16) and conjugated monomers (DC11MAF and C2DC11MAF) at 1, 2, or 3% [82], base bone cement-Osteopal V modified with castor oil and linoleic acid [86], 2-hydroxyethyl methacrylate (HEMA) and isobutyl methacrylate (IBMA) at 2, 3, and 5% [89], and MUPB (monomer methacryloyloxyundecylpyridinium bromide) [101] showed cytotoxic effect from moderately cytotoxic (cell viability ranging from 25 to 50%) to severely cytotoxic (cell viability lower than 25%).

Only three studies were reported about the genotoxicity test where the exposure of occupation time did not show difference from patients without continuous exposure or cell culture [98, 105]. It is necessary to include genotoxicity assay for further investigations of potential biomaterials in dental practice.

Several studies have been carried out at short- or long- term implantation with PMMA enriched or coated with different materials as base bone cement-osteopal V modified with castor oil and linoleic acid in rats [86], coating with titanium dioxide (TiO<sup>2</sup> ) nanoparticles for hamster oral mucosa irritation and guinea pig skin sensitization and intracutaneous rabbit implantation [106], enriched with CPC in rats [90], scaffolds of electrospinning using polycaprolactone (PCL) implantation in rats [94], no toxic effect or histological findings were observed, even a regeneration was perceived. By contrast, the use of PMMA-PEI nanoparticles injected in mice induces significant toxicity by the detection of protein levels in liver tissue.

Acrylic resin cytotoxicity is associated with the presence of residual monomer in the polymerization process. The monomers change cell morphology and function that can reduce their viability. Since acrylic resins are widely used in dental practice, an acceptable biocompatibility is desirable. Considering that the majority of studies reported acrylic resin toxicity responses, further studies with different assessment methods are necessary for the development of biocompatible materials.

In summary, there exist different methods to evaluate acrylic resin cytotoxicity, genotoxicity, and short- or long-term implantation with the MTT method and histological evaluation being the most common tests. In conclusion, there is no non-cytotoxic acrylic resin evidently available in the dental market. Regarding the methods of polymerization, the autopolymerized resin is more cytotoxic and toxic than heat-polymerized resin. The cytotoxic and toxicity effect is dose dependent and is directly correlated with the residual amount of monomer leachable and induce the inflammatory reactions of tissues in contact with the acrylic resin. It is suggested that a water or ethanol bath after polymerization of acrylic resin could decrease the cytotoxic and toxicity activity against oral cells and tissue.
