Evaluation of Regular Market Ethyl Cyanoacrylate Cytotoxicity for Human Gingival Fibroblasts and Osteoblasts

Abstract

Background:

The aim of this study was to evaluate the cytotoxicity of cyanoacrylate adhesives in an indirect contact assay in human gingival fibroblast (FGH) and oral osteoblasts (GO) lineages.

Methods:

Cover glasses were glued with adhesives following the ISO 10993-2012 protocol. The groups were: C (control with cells and regular Dulbecco Modified Eagle Medium; LC (liquid ethyl-cyanoacrylate); GC (ethyl-cyanoacrylate gel); EGC (easy gel [ethyl-cyanoacrylate]); and D (Dermabond [octyl-cyanoacrylate]). Each cell linage was plated in the sixth passage using 10⁴ cells. Cell viability was measured by the MTT test at 24, 48, 72, and 96 hours. Data were analyzed by two-way analysis of variance complemented by the Tukey test, with p < 0.05 being significant.

Results:

Dermabond stimulated osteoblast viability at 72 h (p < 0.05). All other groups were similar to the control cells (p > 0.05). For the fibroblasts, there was no difference in the groups, including the control except that EGC was cytotoxic for these cells (p < 0.05).

Conclusions:

Ethyl-cyanoacrylate gel and liquid forms available on the general chemical market were not cytotoxic for oral osteoblasts and fibroblasts in most cases. However, the easy gel form was cytotoxic for fibroblasts.

Cyanoacrylate (CA) is an acrylic bonded compound synthesized in 1949, whose chemical and adhesive properties were characterized in 1959. It is a versatile adhesive, being easily transportable and aiding in public health for treatment in remote communities [1]. It has hemostatic and anti-inflammatory properties and high adhesiveness for humid substrates, besides being biodegradable, bacteriostatic, and usually exhibiting fast polymerization.

In vivo research showed positive action and no toxicity of CAs as hemostatic agents, topical dressing, and bonders for soft tissues, repair of blood vessels and nerves, osteosynthesis of fractures [2], and closure of perforations in Schneider's membrane during maxillary sinus lift procedures [3]. There also are reports of numerous applications of CA as a substitute for sutures [4]. Recently, CA was used with success in cases of bronchial artery embolization in patients with recurrent hemoptysis and reinforcement of the staple line in laparoscopic sleeve gastrectomy. In dentistry, CA can maintain resorbable membranes in position during and after surgical procedures [5] and act as an auxiliary method in rubber dam fixation [6], intra-oral synthesis in surgical grafts [7], suture of incisions of the face [8], and even fixation of the semilunar approach in root coverage [9] and closing sockets after extraction [10].

The cytotoxicity of CA is controversial, especially when tested in cell culture. The cytotoxicity in vitro and in vivo is linked to formaldehyde release as a product of hydrolytic degradation. The larger the lateral chain, the lower the degradation speed and histotoxicity [11]. In vitro, methyl-cyanoacrylate (Three Bond; Diadema, São Paulo, Brazil) is more toxic than ethyl-cyanoacrylate (SuperBonder; Loctite, Itapevi, São Paulo) [12]. The CA for medical use is considered non-toxic (Dermabond^®; Ethicon, Somerville, NJ) and is an octyl-cyanoacrylate that is sterile and widely used in surgical techniques [13].

Several research projects employed the ethyl-cyanoacrylate sold at on the regular market (SuperBonder; Itapevi, São Paulo) in surgical areas aiming at technical facilitation, reduction of operative time, and low cost [14]. This CA did not promote adverse inflammatory reactions when analyzed in vivo [15]. Microbiologic studies in vitro evaluated the biocide activity and demonstrated no contamination with the use of these agents [16]. At the histologic level, it was possible to observe formation of small giant-cell granulomas around the bond without a neutrophil inflammatory infiltrate indicating a local bacterial or chemical injury [17]. An in vitro study with mouse fibroblasts demonstrated progressive, continuous cell growth, similar to that observed with common commercial brands [18]. However, the current literature reports concern about toxicity [19] and hypersensitivity [20] for several CA agents used in pre-clinical and clinical studies [21].

Few studies investigated the potential toxic effects of CA in human cell cultures [22]. In relation to oral cells, one study showed toxicity of a CA superglue for gingival fibroblasts [22] and another that ethyl-cyanoacrylate was compatible with culture of osteoblasts harvested from alveolar bone [12].

Because of the small number of studies on human-derived cells and the controversies in the literature on the effects of CA on human cells, this research evaluated the in vitro toxicity for human oral osteoblasts and gingival fibroblasts of different CA agents sold on the regular market or for medical use.

Material and Methods

After ethical approval, fibroblasts (FGH cell lineage) obtained by primary culture from a fragment of human gingiva were used. Also, osteoblasts of bone granulation (GO lineage) were obtained by primary culture from dental alveolus with 28 days of healing [23,24]. Cells were cultured in Dulbecco Modified Eagle Medium (DMEM) (Sigma Chemical Co, St Louis, MO) containing 10% fetal bovine serum (Cultilab, Campinas, SP, Brazil) supplemented with 1% antibiotic and 0.5% antimycotic solution (Sigma). Cells were maintained at 37°C in an atmosphere containing 5% CO₂ and 95% air.

This study was conducted by the indirect method, by contact of cells with conditioned medium, not direct cyanoacrylate–cell contact. Two cover glasses were glued with different adhesives. The amount of glue used followed the proportion established by ISO 10993-2012 (4 g/mL) for medium conditioning. After bonding, each specimen was weighed in a precision scale (Mettler-Toledo Ind. e Com. Ltda. Tamboré–Barueri, SP) seeking homogeneity of specimens. A proportion of 3.2 g of glue/16 mL of medium was used for each group. One minute was allowed for glue drying; then the specimens were added to the medium, which was conditioned by the glued cover glasses for 24 hours in a cell incubator. The control group received conventional DMEM without medium conditioning.

The study tested a suitable adhesive for medical use (Dermabond; octyl-cyanoacrylate) and three adhesives found in the public market: Ethyl-cyanoacrylate (Superbonder) liquid, gel and easy gel, the latter with modified chemical composition to delay polymerization.

The experimental groups were divided into:

C (control group with cells and regular DMEM);

LC (cyanoacrylate liquid [Super Bonder^®, Loctite Henkel, Brazil; ethyl-cyanoacrylate]);

GC (cyanoacrylate gel [Super Bonder^®, Loctite Henkel; ethyl-cyanoacrylate]);

EGC (cyanoacrylate easy gel [Super Bonder^®, Loctite Henkel; ethyl-cyanoacrylate]);

D (Dermabond^®; Ethicon, Sommerville, NJ; octyl-cyanoacrylate).

When the cells reached the sixth passage, they were seeded in 96-well plates (1.2 × 10⁵/well) with six replicates per group. After 24 hours in an incubator, the culture media were replaced by conditioned media created by the different treatments; thus, the cells had indirect contact with the tested agents. For the control group, the DMEM was refreshed.

Cell proliferation was evaluated at 24, 48, 72, and 96 hours for analysis of the mitochondrial activity of the cells using the MTT reduction method (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide)[25]. The results represent the mean of three repeated assays.

Data on optical density were analyzed on Statistica 11.0 (www.statsoft.com/company) by one-tailed ANOVA for repeated measures complemented by the Tukey test. This test was used because of the normal distribution of data and for comparing all groups versus all times. The study adopted a significance level of p < 0.05.

Results

Table 1 shows the viability of oral osteoblasts after incubation in conditioned medium with different adhesives. In general, viability increased until 72 hours and remained stable until 96 hours, probably because of cell confluence in the wells. The highest optical density values were observed in the D group at 72 and 96 hours and the control and LC groups at 96 hours. Dermabond stimulated cell viability at 72 hours, with significant differences from the other groups (p < 0.05). All groups were similar to the control cells at other periods, demonstrating the absence of cytotoxicity for osteoblasts.

Table 1.

Oral Osteoblasts (GO lineage) Viability (Optical Density) by Group and Time

H	C	D	LC	EGC	GC
24	0.253 ± 0.06^a	0.465 ± 0.09^ab	0.485 ± 0.01^abc	0.449 ± 0.06^ab	0.563 ± 0.12^bcd
48	0.640 ± 0.13^cde	0.712 ± 0.10^cdef	0.775 ± 0.13^defg	0.582 ± 0.08^bcd	0.623 ± 0.09^bcd
72	0.895 ± 0.11^fghi	1.257 ± 0.20^j	0.948 ± 0.17^fghi	0.952 ± 0.08^fghi	0.964 ± 0.13^ghi
96	1.070 ± 0.10^hij	1.094 ± 0.07^hij	1.116 ± 0.07^ij	0.875 ± 0.08^efgh	0.893 ± 0.11^fghi

Different lowercase letters = p < 0.05. Group × period intercept = p = 0.000004.

C = control; D = Dermabond; EGC = easy gel cyanoacrylate; GC = gel cyanoacrylate; LC = liquid cyanoacrylate.

In relation to fibroblasts, Table 2 shows a tendency to increasing cell viability up to 96 hours (p < 0.05). There was no difference between the test groups and the control population with the exception of EGC, for which the optical density values were lower than those of the control (p < 0.05). Easy gel (EGC) impaired cell viability, manifesting cytotoxicity for the fibroblasts.

Table 2.

Gingival Fibroblast (FGH Lineage) Viability (Optical Density) by Group and Period

H	C^ab	D^a	LC^a	EGC^b	GC^ab
24^A	0.083 ± 0.02	0.068 ± 0.03	0.096 ± 0.03	0.085 ± 0.03	0.109 ± 0.05
48^A	0.197 ± 0.07	0.208 ± 0.05	0.197 ± 0.06	0.102 ± 0.04	0.151 ± 0.03
72^B	0.261 ± 0.08	0.365 ± 0.12	0.284 ± 0.07	0.204 ± 0.08	0.212 ± 0.11
96^C	0.751 ± 0.11	0.701 ± 0.35	0.762 ± 0.12	0.513 ± 0.11	0.488 ± 0.25

Different uppercase letters = p < 0.05 for period; different lowercase letters = p < 0.05 for groups. Group × period intercept p = 0.137268.

C = control; D = Dermabond; EGC = easy gel cyanoacrylate; GC = gel cyanoacrylate; LC = liquid cyanoacrylate.

Table 3 shows a comparison between cells. Optical density values were converted to percentage of growth considering the 24-hour period as 100%.

Table 3.

Percent of Viable Cells at Different Times for Human Gingival Fibroblasts and Osteoblasts after Incubation in Conditioned Medium

Time (H)	C	D	LC	EGC	GC
GO
24	100^ab	100^ab	100^ab	100^ab	100^ab
48	236 ± 85^abcde	153 ± 23^abc	159 ± 27^abc	129 ± 18^abc	110 ± 16^abc
72	354 ± 46^cdefg	270 ± 44^abcde	195 ± 36^abcd	212 ± 18^abcde	171 ± 23^abc
96	423 ± 39^defg	235 ± 16^abcde	230 ± 15^abcde	194 ± 18^abcd	158 ± 21^abc

FGH
24	100^ab	100^ab	100^ab	100^ab	100^ab
48	261 ± 98^abcde	305 ± 74^abcde	205 ± 67^abcde	120 ± 48^abc	139 ± 28^abc
72	345 ± 115^bcdef	537 ± 185^fg	296 ± 79^abcdef	239 ± 96^abcde	194 ± 107^abcd
96	994 ± 132ⁱ	1031 ± 525ⁱ	792 ± 118^hi	601 ± 124^gh	447 ± 232^efg

Different letters = p < 0.05.

C = control; D = Dermabond; EGC = easy gel cyanoacrylate; GC = gel cyanoacrylate; LC = liquid cyanoacrylate.

Osteoblast metabolism was slower than that for fibroblasts, as expected. Osteoblasts from the control group had a fourfold increase in viability at the end of 96 hours, whereas in the other groups, viability was approximately twofold higher. For fibroblasts at 96 hours, there was a 10-fold increase in cell viability versus the D and control groups.

The GO lineage, in contact with different forms of cyanoacrylates, presented percentages of cell viability similar to the control cells at all times with the exception of GC at 96 hours, which presented reduced cell viability in relation to the control cultures (p < 0.05). The FGH lineage presented differences only at 96 hours. Both GC and EGC presented a statistically significant reduction in the percentage of cell viability in relation to D and control cells. The LC, D, and control groups presented the highest viability percentages, with no differences among them.

Discussion

The toxicity of CA agents was evaluated in cultures of human oral osteoblasts and fibroblasts. The CA of medical-use Dermabond (2-octyl-cyanoacrylate) increased the viability of osteoblasts. The CA easy gel, with agents to delay polymerization, was more toxic for fibroblasts, probably because of the chemicals added to the formula. The other forms of ethyl-cyanoacrylate liquid and gel demonstrated biocompatibility, and the cells behaved in a manner similar to the control group. It should be highlighted that, except for Dermabond, the tested products are adhesives sold on the open market. Despite the off-label application in health areas, such products exhibit low cost, making them an option for use in the poorest locations.

When not polymerized, CAs are highly toxic [26]. The size of the lateral chain of CA is related to its biocompatibility, and the short-chain CA (ethyl- and methyl-cyanoacrylates) demonstrated more toxicity [27]. Long-chain adhesives are less toxic because they release smaller amounts of formaldehyde [19]. Besides the chemical composition of CA, it should be considered that each cell lineage can react differently to the bonder. The butyl-cyanoacrylate CA does not reduce the viability of L929 cells [28]; however, that same CA showed toxic effects on 3T3 cells [29]. The same was observed for octyl-cyanoacrylate, which affected the cycle of cells derived from human placenta but did not demonstrate toxicity for L929 cells [26].

Controversial results exist in relation to the toxicity of ethyl-cyanoacrylate adhesives in in vitro research. Studies have shown toxic effects [30], reducing the cellular protein content [11]. Conversely, their effects were similar to octyl-cyanoacrylate for medical use [18,27], displaying good results in the viability of osteoblasts [12], thus being recommended for clinical use [31]. Studies tested the effects of CA on oral fibroblasts [22] and osteoblasts [12]. The ethyl-cyanoacrylate adhesives for common use release substances that are toxic for fibroblasts in long-term follow-up (two weeks) [22]. In osteoblasts, ethyl-cyanoacrylate was less toxic than methyl-cyanoacrylate, reaching numbers similar to the control cultures, thus confirming biocompatibility [12].

When adhesives were tested in cell culture, the results suggested that, when the amount of CA used directly is reduced by diluting it or using indirect contact methods, toxicity also is reduced [26,32 -34]. When cells were put in direct contact with a bonder, in some cases, good results were not observed for L929 fibroblasts [11], but others exhibited biocompatibility for human osteoblasts [12] and also for L929 fibroblasts [26]. There also are reports of transitory toxicity for neuroblastoma cells [35]. The present study employed the indirect method of cytotoxicity evaluation by the conditioned medium [36]. The medium was conditioned for cover glasses agglutinated with different adhesives for 24 hours in appropriate culture conditions. Any toxic products resulting from adhesive polymerization would be released into the culture medium yet be diluted. This suggests that, when applied to patients, the products of CA polymerization can be eliminated quickly by the organism by inflammatory exudate or buffering system. Therefore, the indirect method was able to mimic that clinical condition. Additionally, utilization of an ISO norm allows standardization and comparison between studies. Following the standards foreseen in ISO 10993 norm (4 g/mL), the present study achieved a considerable dilution of toxic products. The concentration used in the present study was similar to that in other studies that used different dilutions of CA in indirect contact assays [21,31,33]. The results showed no alteration in material toxicity in low dilutions, suggesting the value of the clinical use of the smallest amount possible to avoid adverse effects [34]. The MTT assay demonstrates great sensitivity to cytotoxic analysis compared with other methods [37].

The safety of commonly available CA products for clinical use can be based on animal studies. Histologic analyses demonstrate absence of neutrophil reaction, indicating that there was no chemical or bacterial injury. After seven days of healing, CA was surrounded by macrophages forming giant-cell granulomas, similar to what is observed around tattoo pigments [17].

A study employing L929 cells and indirect contact revealed that ethyl-cyanoacrylate in fluid form was more toxic, with a slightly lower degree of toxicity in gel form [38]. Such results differ from those of the present study, in which both liquid and gel ethyl-cyanoacrylate presented favorable results concerning cell viability, mainly for osteoblasts (GO). Conversely, when the gel with delayed polymerization was used in the CEG group, the fibroblasts (FGH) exhibited significantly altered viability, corroborating the previously mentioned results. Other studies employing cover glasses for indirect contact between CA and cells (NIH3T3) demonstrated that ethyl-cyanoacrylate liquid caused effects similar to a sterile CA for medical use (Histoacryl/butyl-cyanoacrylate) [18,35]. The results were much like those of the present study, as the non-sterile ethyl-cyanoacrylate liquid (CL group) produced results similar to the Dermabond group (octyl-cyanoacrylate) in both cell lineages.

The Dermabond/octyl cyanoacrylate group presented the best rates of cell viability, in agreement with previous studies with the same contact model and cell lineages HS68 and L929 [39], confirming the safety and effectiveness for the employed cells. However, such results were not observed in cells derived from human placenta [40].

To the best of our knowledge, this is the first project to use human-derived bone cells in an indirect cytotoxicity assay with CAs and using an ISO norm, a standard, validated assay to mimic clinical scenarios. Studies evaluating the in vitro toxicity of these agents in human-derived cells and employing standard norms are scarce in the literature [22]. We employed cover glasses because we believe that those substrates are less subject to variations during medium conditioning, facilitating the creation of good specimens [18]. That standardization allowed comparison of different glues in two cell lineages.

Our results corroborate the intention of clinical use of agents derived from ethyl cyanoacrylate available in the common market [31]. Although being an off-label application of adhesives, this study revealed similarity of results with CA of medical use regarding cell viability. Even though the agents for conventional use are not sterile, there are reports of countless applications and purposes for those agents in the literature [9, 17].

The pre-clinical research has importance in allowing comparison and analysis of the toxicity of adhesives. Formulations will not always present the same results, as they have different compositions around the world; e.g. the cyanoacrylate easy gel with delayed polymerization used in this study. Even though the main agents are similar, the additives in each formula modify or carry substances with products that can generate or cause noxious interactions biologically, and the indirect way assay employed in our study was sensitive enough to show these effects. The conventional liquid (ethyl-cyanoacrylate) achieved results similar to a CA for medical use. Common adhesives were not cytotoxic for cells, showing biocompatibility similar to those manufactured for medical use. In addition, they are inexpensive and can be used for a variety of procedures in low-income countries. Our results also may support the findings of future studies employing scaffolds or other three-dimensional models that is necessarily bonded to different substrates.

Conclusion

The octyl-cyanoacrylate formulated for medical use stimulated the viability of oral osteoblasts, whereas ethyl-cyanoacrylate easy gel was cytotoxic for oral fibroblasts. The other two forms of ethyl-cyanoacrylate (gel and liquid) available on the general market were not toxic to either type of cell.

Footnotes

Author Disclosure Statement

This work was supported by the Pró-reitoria de pesquisa da Universidade de São Paulo (grant numbers RUSP #2014-322) and also in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brasil (CAPES) - Finance Code 001.

This research involved human participants. All procedures were performed in accordance with the ethical standards of the institutional Ethical Committee on Human Research of Bauru School of Dentistry–University of São Paulo (#086/2011) and/or the national research committee and with the 1964 Helsinki Declaration and its later amendments or comparable ethical standards.

Informed consent was obtained from all individual participants included in the study.

No competing financial interests exist for any of the authors.

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