Cytoprotective effects of Hyaluronic Acid and Carbomer 934P in Ocular Surface Epithelial Cells

Abstract

purpose. To investigate in vitro the cell toxicity and antioxidant effects of two major tear substitutes, hyaluronic acid and a widely used carbomer, with and without preservative.

methods. Chang conjunctival cells were treated with different concentrations of unpreserved or preserved carbomer 934P (0.03% and 0.3%), unpreserved or preserved hyaluronic acid (0.018% and 0.18%), and benzalkonium chloride (BAC 0.0005% and 0.005%) for 15 minutes or for 15 minutes with 24 hours of cell recovery, according to previously validated methods. Microplate cold light cytofluorometry was performed to evaluate cell viability (neutral red test), chromatin condensation (Hoechst 33342 test), and reactive oxygen species (ROS) production (dichlorofluorescein diacetate and hydroethidine tests). Confocal microscopy was used to explore morphologic changes.

results. No alterations were found with unpreserved and preserved hyaluronic acid at all concentrations and times tested. A decrease in cell viability with chromatin condensation appeared with 0.3% preserved carbomer 934P at the two times tested. This cytotoxicity, however, was significantly less than that observed with BAC alone, although the same concentrations of preservative were used. Unpreserved carbomer 934P induced no modification of cell viability after 15 minutes but a significant decrease in chromatin condensation, reversible after 24 hours of cell recovery, when a delayed decrease in cell viability was observed. Production of reactive oxygen species (ROS) decreased with the four formulations of tear substitutes tested at their usual concentrations, whereas a significant production of ROS occurred with BAC.

conclusions. These two ophthalmic hydrogels have no cytotoxicity but possess antioxidant properties and tend to reduce the toxic effects of preservatives. These results may allow use of hydrogels, not only in dry eye but also in ocular surface disorders involving oxidative stress and in ophthalmic drug therapy to improve ocular tolerance.

The ocular surface is covered by tear film, which provides immune protection and has metabolic and optic roles. ¹ Abnormalities in lipidic, aqueous, or mucous layers of the tear film or the corneoconjunctival epithelium are responsible for various diseases, mainly dry-eye syndrome. ² These diseases require long-term instillation of artificial tears to maintain the integrity of the ocular surface epithelium. One of the major problems encountered with solutions is the rapid and extensive elimination of drugs from the precorneal lacrimal fluid by solution drainage or lacrimation. ³ Consequently, the ocular residence time of conventional solutions is limited to a few minutes, and the overall absorption of a topically applied drug is limited to 1% to 10%. ⁴ One of the common methods of optimizing prolonged precorneal residence time is the use of polymers to increase solution viscosity. In comparison with traditional formulations, this system has the following advantages: increased contact time, ⁵ prolonged drug release, ^{6 7} reduction of systemic drug effects, ⁸ and better compliance by the patient. 

Many artificial tear preparations are now commercially available. However, most of them have to be used frequently to be effective, and preservatives are usually added to ophthalmic solutions to prevent contamination. Nevertheless, benzalkonium chloride (BAC), the most common preservative that provides antimicrobial effectiveness, is known to disrupt the tear film ⁹ and to impair epithelial cell membranes. Furthermore, an increasing number of studies in both animals ^{10 11 12} and humans ^{13 14} have shown that preservatives are liable to damage the ocular surface, and it is thus suspected that they impair the local tolerance of eye drops. ^{15 16} Paradoxically, the well-known toxic effects of BAC have been used to enhance penetration of the principal drug, ¹⁷ because of the compromise of the epithelial barrier. 

Preliminary in vitro studies have demonstrated that unpreserved tear substitutes do not induce any toxicity in conjunctival cells, ¹⁸ whereas BAC induces arrest of cellular growth and cell death at a concentration as low as 0.0001%. ^{19 20}  

The purpose of the present study was to determine whether two hydrogels widely used as tear substitutes, carbomer 934P and hyaluronic acid, could interact with BAC to reduce the toxic effects of this quaternary cationic surfactant on conjunctival epithelium; to examine whether sodium hyaluronate would provide any advantage over synthetic carbomers; and to evaluate the eventual antioxidant effects of hyaluronic acid and carbomer 934P. We used a human continuous conjunctival cell line as in previous in vitro toxicology studies. ^{19 20 21} We performed cold light cytofluorometry, ²² which had been validated in previous studies, ^{16 20 23 24} to evaluate cell viability, DNA condensation, and ROS production and confocal microscopy to assess the morphologic patterns of cells. 

Materials and Methods

Conjunctival Cell Line

A human conjunctival cell line (Wong Kilbourne derivative of Chang conjunctiva, clone 1-5c-4, CCL–20.2; American Type Culture Collection [ATCC], Manassas, VA) was cultured under standard conditions (humidified atmosphere of 5% CO₂ at 37°C) in Dulbecco’s minimum essential medium (DMEM) (Eurobio, Les Ulis, France) supplemented with 10% fetal bovine serum (Dominique Dutscher, Brumath, France), 1% glutamine (Eurobio), 0.1% ampicillin (Panpharma, Fougères, France), and 2% kanamycin (Bristol-Myers Squibb, Paris, France), as previously described. ²⁰ Normal culture development was assessed daily by phase-contrast microscopy. Confluent cultures were removed by gentle trypsin incubation, and cells were counted. They were then seeded into 96-well culture plates for microtitration analysis (5000 cells per well; Nunc, Roskild, Denmark). After subconfluence had been attained (culture surface covering nearly 70%), cells were exposed to the different formulations. 

Cell Treatments

Unpreserved hyaluronic acid (HA BAC⁻, Vismed; Transphyto, Clermont-Ferrand, France), preserved hyaluronic acid (HA BAC+ with 0.005% BAC; Transphyto), BAC (Transphyto), unpreserved carbomer 934P (Gel-larmes; Transphyto) (carbomer 934P BAC⁻) and preserved carbomer 934P (carbomer 934P BAC⁺ with 0.005% BAC, Gel-larmes; Transphyto) were tested. These five drugs were evaluated at their usual commercial concentrations (0.18% for unpreserved and preserved hyaluronic acid, 0.005% for BAC, 0.3% for unpreserved and preserved carbomer 934P) and after a 1:10 dilution in culture medium. The complete culture medium was used as a negative control. 

In the present study two incubation times were therefore applied to control and treated cells: 15 minutes of treatment and 15 minutes of treatment with 24 hours of cell recovery in normal culture medium, as in our previous studies. ^{16 20}  

Experimental Procedures

Microplate Cold Light Fluorometry.

Experiments were performed by microplate cold light fluorometry, as previously validated ²⁰ (Fluorolite 1000 Dynex Thermobioanalysis microplate fluorometer ²² ; Dynex, Issy-Les-Moulineaux, France. According to the recommendations of the European Center for the Validation of Alternative Methods (ECVAM) and to previously validated methods in the Chang cell line, ^{15 18} three cellular markers were evaluated: cellular viability, cellular proliferation, and ROS production. 

Cellular Viability Assay.

Briefly, cellular viability was evaluated with a neutral red (Fluka, Ronkonkoma, NY) test with fluorometric detection (excitation, 535 nm; emission, 600 nm). Neutral red was used at 50 μg/mL after exposure of cells to the different drugs. ²⁰  

Chromatin Condensation Assay.

Hoechst 33342 (Molecular Probes, Eugene, OR) is an intercalating dye which allows the determination of the total chromatin quantity variations and the degree of chromatin condensation. ²⁵ It specifically reacts with the DNA at the adenine and thymidine levels. This probe was used on cells at a final concentration of 10 μg/mL (excitation, 360 nm; emission, 450 nm). Propidium iodide (Roche Diagnostics Corp., Meylan, France) at 0.5 mg/mL was added to the Hoechst solution to control necrotic cells (this DNA probe reacts by intercalation and does not allow Hoechst fixation by necrotic cells), as previously validated. ¹⁶ Supravital uptake of Hoechst 33342 combined with exclusion of propidium iodide was proposed as an assay for apoptosis. ²⁵  

ROS Assays.

O₂ ^⨪ was detected using hydroethidine (HE) (Molecular Probes) at 5 μM (excitation, 485 nm; emission, 600 nm). ^{20 26} HE is oxidized to the fluorescent ethidium cation by O₂ ^⨪, allowing the cation to bind to nuclear DNA with an extensive fluorescent enhancement. ²⁶  

ROS production was detected with the DCFH-DA (2′,7′-dichlorofluorescein diacetate; Molecular Probes) dye. This probe is a sensitive peroxide-specific fluorescence marker used on cells at 20 μM (excitation, 490 nm; emission, 535 nm). ^{20 24} Once inside the cell, it is cleaved by endogenous esterases and can no longer pass out of the cell. The de-esterified product becomes the fluorescent compound 2′,7′-dichlorofluorescein after oxidation by ROS. The fluorescent signal detected has been demonstrated to be proportional to ROS production. ²⁷  

Statistical Analyses

In all experiments, the background fluorescence was determined in wells without cells but containing the dye solution. Fluorescence was deduced from all control and treated wells. 

Microplate cold light cytofluorometry results were obtained in fluorescence units and were expressed as a percentage of the control. Wells containing cells with complete culture medium but without any treatment were used as the control. Each drug concentration was tested in six wells, and each experiment was realized in triplicate. Statistical comparisons were performed with a Mann-Whitney test at a 0.05 level of significance (Statview IV for Windows; Abacus, Berkeley, CA). 

Immunocytology

In parallel, standard immunofluorescence was performed to assess morphologic patterns of cells. Cells were cultured on slides (Laboratory-tek II chambered coverglass; Nalge Nunc International, Napierville, IL) and treated with the hydrogels tested for 15 minutes. They were washed with PBS and fixed 10 minutes with 95% ethanol in PBS at −20°C. Fluorescent phalloidin (200 U/mL, Alexa 488; Molecular Probes) was then added to detect F-actin. After 30 minutes of incubation, cells were washed in PBS. Propidium iodide was added to mark cell nuclei before examination with a confocal epifluorescence microscope (E800, PCM 2000; Nikon, Tokyo, Japan). 

Results

Cellular Viability Evaluation

Compared with the control, after 15 minutes of treatment and at their usual concentrations (Fig. 1A) , unpreserved hyaluronic acid, preserved hyaluronic acid, and unpreserved carbomer 934P did not modify cellular viability (mean fluorescence, respectively, 105%, 107%, and 93% of the control, nonsignificant), whereas preserved carbomer 934P and BAC induced significant cellular damage (mean fluorescence, respectively, 78% and 21% of the control, P = 0.0149 and P = 0.0002 compared with control; Table 1 ). No significant differences were found between unpreserved formulations, whereas preserved hyaluronic acid was found to be significantly less cytotoxic than preserved carbomer (P < 0.0001). With the 1:10 dilution (Fig. 1B) , a slight but significant increase in cell viability was observed with both unpreserved and preserved carbomer 934P (mean fluorescence, respectively, 123% and 116% of the control, P = 0.0122 and P = 0.0392 compared with control), whereas unpreserved hyaluronic acid, preserved hyaluronic acid, and BAC did not modify this parameter (mean fluorescence, respectively, 104%, 106%, and 95% of the control, nonsignificant; Table 1 ). 

After 24 hours of cell recovery (Fig. 2 ; Table 2 ), a significant decrease was observed with unpreserved and preserved carbomer 934P (mean fluorescence, respectively, 65% and 57% of the control, P = 0.0034 and P = 0.002 compared with control) tested at their usual concentrations (Fig. 2A) , whereas no alteration was found with unpreserved and preserved hyaluronic acid (mean fluorescence, respectively, 92% and 94% of the control, nonsignificant compared with control). Unpreserved and preserved hyaluronic acid induced significantly less cytotoxicity than unpreserved and preserved carbomer (P < 0.0001). BAC still induced a significant decrease in cell viability (mean fluorescence: 26% of the control, P = 0.0004 compared with control). With the 1:10 dilution (Fig. 2B) , unpreserved and preserved carbomer 934P and unpreserved and preserved hyaluronic acid did not modify cellular viability (mean fluorescence, respectively, 102%, 107%, 94%, and 101% of the control, nonsignificant), whereas BAC induced a significant decrease in this parameter (mean fluorescence: 86% of the control, P = 0.04 compared with control). 

After 15 minutes of treatment or 15 minutes of treatment with 24 hours of cell recovery (Figs. 1A 2A) , the four hydrogels tested at their usual concentrations were found to be significantly less toxic than BAC alone (P < 0.0001 compared with BAC), although the same concentrations of the preservative were used. 

DNA Condensation Evaluation

Compared with control, after 15 minutes of treatment and at its usual concentrations (Fig. 1A) , unpreserved carbomer 934P induced a significant decrease in Hoechst fluorescence (mean fluorescence: 62% of the control, P = 0.0025 compared with control). Preserved carbomer 934P and BAC induced a significant increase in Hoechst fluorescence (mean fluorescence, respectively, 189% and 381% of the control, P = 0.0007 and P < 0.0001 compared with control), whereas no modification was observed with unpreserved and preserved hyaluronic acid (mean fluorescence, respectively, 108% and 119% of the control, nonsignificant, P = 0.1856 and P = 0.0536 compared with control). With the 1:10 dilution (Fig. 1B) , chromatin condensation was observed only with BAC (mean fluorescence: 138% of the control, P = 0.0025 compared with control). 

After 24 hours of cell recovery and at their usual concentrations (Fig. 2A) , no modification of Hoechst fluorescence was observed with unpreserved and preserved hyaluronic acid (mean fluorescence, respectively, 92% and 93% of the control). A significant decrease in Hoechst fluorescence was found with unpreserved carbomer (mean fluorescence: 83% of the control, P = 0.0235 compared with control), whereas a significant increase was shown with preserved carbomer and with BAC (mean fluorescence, respectively, 123% and 264% of the control, P = 0.013 and P < 0.0001 compared with control). With the 1:10 dilution (Fig. 2B) , Hoechst fluorescence was significantly increased only with BAC (mean fluorescence, 150% of the control, P = 0.0011 compared with control). 

After 15 minutes of treatment as well as 15 minutes and 24 hours of cell recovery (Figs. 1 2) , unpreserved and preserved hyaluronic acid and unpreserved and preserved carbomer 934P induced a significant decrease in Hoechst fluorescence compared with BAC (P < 0.0001 compared with BAC for all the concentrations tested). Preserved carbomer 934P and preserved hyaluronic acid induced less chromatin condensation than BAC, even though the same concentrations were used. No significant variations in Hoechst fluorescence were found between unpreserved and preserved hyaluronic acid, whereas preserved carbomer at 0.3% induced a more significant increase in Hoechst fluorescence than unpreserved carbomer at 0.3% (P < 0.0001 compared with unpreserved carbomer at both 15 minutes and 15 minutes followed by 24 hours of cell recovery). 

O₂ ^⨪ Production with the HE Test

Compared with control, BAC induced a concentration-dependent O₂ ^⨪ synthesis (mean fluorescence, respectively, 118% and 142% with BAC 0.0005% and 0.005%, P = 0.0381 and P = 0.0019 compared with control). At their usual concentrations, unpreserved and preserved hyaluronic acid and unpreserved and preserved carbomer 934P induced a significant decrease in O₂ ^⨪ production (mean fluorescence, respectively, 21%, 33%, 21%, and 24% of the control, P = 0.0003, P = 0.0005, P = 0.0003, and P = 0.0003 compared with control; Fig. 1A , Table 1 ), whereas no modification was observed after a 1:10 dilution (Fig. 1B ; Table 1 ). 

Compared with BAC, a significant decrease in fluorescence was observed with the four hydrogels at all the concentrations tested (P < 0.0001 compared with BAC, despite use of the same concentrations of BAC in preserved hydrogels and BAC solutions, Fig. 1A ). 

Furthermore, unpreserved hyaluronic acid at 0.18% induced a significant decrease in HE fluorescence compared with preserved formulations (P = 0.0325 compared with preserved hyaluronic acid), whereas there was no significant difference between unpreserved and preserved carbomer (Fig. 1A) . 

H₂O₂ Production with DCFH-DA Test

Compared with control, hydroperoxide production was increased only with BAC at 0.005% and 0.0005% (mean fluorescence, respectively, 165% and 195% of the control, P = 0.0006 and P = 0.0002 compared with control). A significant decrease in fluorescence was observed with unpreserved and preserved hyaluronic acid and unpreserved and preserved carbomer 934P tested at their usual concentrations (mean fluorescence, respectively, 14%, 66%, 30%, and 56% of the control, P = 0.0003, P = 0.003, P = 0.0005 and P = 0.0013 compared with control; Fig. 1A , Table 1 ), whereas no modification was observed after a 1:10 dilution (Fig. 1B , Table 1 ). 

Compared with BAC, a significant decrease in fluorescence was observed with the four hydrogels at all the concentrations tested (P < 0.0001 compared with BAC, although tested at the same concentrations). Furthermore, unpreserved carbomer and unpreserved hyaluronic acid induced a significant decrease in DCFH-DA fluorescence compared with preserved formulations (P = 0.0002 compared with preserved carbomer and P < 0.0001 compared with preserved hyaluronic acid, Fig. 1A ). 

Morphologic Changes

A concentration-dependent cell shrinkage was observed after a treatment with BAC, whereas no morphologic change was observed with hyaluronic acid, carbomer 934P or preserved hydrogels. These results confirm that unpreserved hydrogels have no toxic effects in vitro and reveal that preserved formulations do not alter cell morphology, suggesting a protective effect of hydrogels against BAC toxicity (Fig. 3) . 

Discussion

Hydrogels are defined as polymers endowed with the ability to swell in water or aqueous solvents and induce a liquid–gel transition. ²⁸ Currently, two groups of hydrogels are distinguished: preformed (celluloses, polyvinyl alcohol, hyaluronic acid, carbomers) and in situ forming gels (gellan gum, poloxamer, cellulose acetate phthalate latex). The advantages claimed for these commercially available products are good tolerance, prolonged contact time on the ocular surface, and good miscibility with the lacrimal fluid. ^{29 30 31} They improve bioavailability and decrease the side effects induced by systemic absorption of topically applied ophthalmic drugs. 

Initial studies, in the rabbit eye (Draize test) showed that carbopol-940 and hydroxypropyl methyl cellulose do not exhibit the potential for ocular irritation. ³²  

However, only a few toxicology studies have been performed with gel formulations to analyze the putative toxic effects of the formulations. Most of them were realized on corneal cells (SIRC). Carboxy methyl cellulose artificial tears have been found to be less toxic than carbomer gel formulations. ³³ Dilute preparations of sodium hyaluronate have shown only mild reduction in the release of plasminogen activator, suggesting absence of cytotoxicity. ³⁴ Furthermore, it has been demonstrated that unpreserved hydrogels do not induce any toxicity in conjunctival cells. ¹⁸ Many hydrogels are now available, and preservatives are often added to prevent contamination. BAC is widely used in ophthalmic solutions for antimicrobial preservation. Numerous clinical and biological side effects of surfactant preservatives (BAC) have been described, such as ocular irritation, punctate keratitis, gray corneal epithelial haze, decreased corneal epithelial microvilli, and cytotoxicity in corneal and conjunctival epithelial cells. ^{11 12 20}  

Our study confirmed the absence of cytotoxicity of unpreserved carbomer 934P at 0.3% after a 15-minute treatment, compared with control, whereas preserved carbomer 934P induced a significant decrease in cell viability. This decrease was significantly lower than that observed with BAC, even though the concentrations were the same, suggesting a protective effect of carbomer 934P. After 24 hours of cell recovery, a significant decrease in cell viability was found with these two carbomers, compared with control, without any difference between the two formulations. This decrease may be attributed to the viscosity of the solutions, which was higher with carbomer (viscosity 13,000 centipoises [cP]) with carbomer and 10 cP with sodium hyaluronate). Chromatin condensation was modified for the two times tested, and a significant decrease in Hoechst fluorescence was observed with unpreserved carbomer at 0.3%, whereas an increase was shown with preserved carbomer at 0.3%, significantly lower than that observed with BAC. The Hoechst fluorescence increase observed after 24 hours of cell recovery was less than that observed after 15 minutes, suggesting reversible toxicity. The possible protective effect of carbomer against BAC toxicity may be a matter of preventing exposure of the ocular surface to the BAC, rather than a specific cellular effect. 

Concerning preserved and unpreserved sodium hyaluronate, no cytotoxicity was observed either after 15 minutes of treatment or after 24 hours of cell recovery, suggesting an interaction between hyaluronate and BAC that induces a reduction of the toxic effects of this preservative on conjunctival cells. Hyaluronic acid appeared to be better tolerated than carbomer 934P, which induced a decrease in cell viability after 24 hours of cell recovery. Furthermore, a significant decrease was shown in superoxide anion production. This production was previously correlated with apoptosis. ²⁰ These antioxidant properties were found not only with sodium hyaluronate but also with preserved and unpreserved carbomer 934P. 

These results were in accordance with clinical results, which have shown that treatment of patients with sodium hyaluronate for 2 years had no adverse effects ³⁵ and also with a previous study performed in a chick experimental model, ³⁶ in which a combination of sodium hyaluronate 0.1% with BAC 0.01% effectively reduced the toxic effects of preservatives. More recently, it was shown in bovine conjunctival epithelial cells that 0.15% hyaluronate associated with 0.005% BAC did not induce any morphologic or functional alteration, although a slow decrease in transepithelial electrical resistance was observed (but significantly less important than with BAC alone). ³⁷  

A possible explanation may be that ionic attraction between the positive charge of BAC and the negative charge of polyanionic polymers (carboxymethyl cellulose, carbomers or sodium hyaluronate) neutralizes the toxic effect caused by the cationic charge of quaternary ammoniums to the conjunctival and the corneal epithelia. It is also possible that BAC molecules penetrate into the spongelike domain of sodium hyaluronate and disperse within it. Another hypothesis is that the binding of quaternary ammonium cationic preservatives to anionic polymers may shift the binding equilibrium away from the interaction with cell membranes, thus reducing the cytotoxic effects. This binding of preservatives to hydrogels may be influenced by such factors as molecular properties of the preservative and also the free concentration of preservative in equilibrium with hydrogel molecules. 

Furthermore, the ultrastructural studies performed have shown that sodium hyaluronate protects the corneal epithelium against dryness better than hydroxy ethyl cellulose or phosphate-buffered saline. ³⁵ These results are in agreement with those in clinical trials ³⁸ in which various protective solutions were compared during cataract surgery. The protective effects of sodium hyaluronate may be attributed to its special spongelike structure of polysaccharide chains with trapped water. It is assumed that these water molecules are slowly released from the sodium hyaluronate solution and thus provide a wetting medium for the epithelium that is therefore better protected. 

In clinical studies of patients with dry eye symptoms, sodium hyaluronate has been shown to increase tear film stability and reduce subjective symptoms, such as grittiness and burning. ^{39 40} The clinical success of several viscoelastic solutions used as tear substitutes has been corroborated by the absence of cytotoxicity. Furthermore, sodium hyaluronate coats the corneal epithelium for at least 1 hour. ⁴¹  

In conclusion, experimental and epidemiologic studies show that ophthalmic preformed hydrogels are well tolerated. The present study suggests that hyaluronic acid and carbomer 934P have no toxic effects in vitro but possess antioxidant properties and that these two hydrogels in combination with BAC reduce the toxic effects of the preservative. Furthermore, tear substitutes containing hyaluronic acid may be more protective of the ocular surface. In the future, a considerable improvement of ocular tolerance may be obtained by adding low concentrations of hydrogels to ophthalmic drugs. However, caution must be used when extrapolating these in vitro findings to the clinical setting. Further studies of hydrogel effects on the human ocular surface are needed before a final conclusion can be reached. 

 Submitted for publication November 7, 2001; revised April 30, 2002; accepted May 31, 2002.

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Figure 1.

 

Cellular viability (neutral red test), DNA condensation (Hoechst 33342 test), O₂ ^⨪ production (HE test), and H₂O₂ production (dichlorofluorescein diacetate [DCFH-DA] test) evaluations after 15 minutes of treatment with carbomer 934P, hyaluronic acid, or BAC. Results are expressed as a percentages of control values (solid horizontal line). (A) Hydrogels tested at their usual concentrations (unpreserved and preserved HA, 0.18%; unpreserved and preserved carbomer, 0.3%) and BAC tested at 0.005%. (B) Hydrogels and BAC tested after a 1:10 dilution in normal culture medium. *P < 0.05; **P < 0.01, and ***P <0.001 compared with the control.

Table 1.

 

Statistical Analysis Summary after 15 Minutes of Treatment

	Carbomer 934P/BAC−			Carbomer 934P/BAC+			Hyaluronic acid/BAC−			Hyaluronic acid/BAC+			BAC
		0.3*	0.03	0.3	0.03	0.18	0.018	0.18	0.018	5 × 10−3	5 × 10−4
Cellular viability
Compared with control	NS	0.0122↑	0.0149↓	0.0392↑	NS	NS	NS	NS	0.0002↓	NS
Compared with BAC	<0.0001↑	<0.001↑	<0.0001↑	<0.001↑	<0.0001↑	NS	<0.0001↑	NS	—	—
DNA condensation
Compared with control	0.0025↓	NS	0.0007↑	NS	NS	NS	NS	NS	<0.0001↑	0.0025↑
Compared with BAC	<0.0001↓	<0.0001↓	<0.0001↓	<0.0001↓	<0.0001↓	<0.0001↓	<0.0001↓	<0.0001↓	—	—
O2 ⨪ production
Compared with control	0.0003↓	NS	0.0003	NS	0.0003↓	NS	0.0005↓	NS	0.0019↑	0.0381↑
Compared with BAC	<0.0001↓	<0.0001↓	<0.0001↓	<0.0001↓	<0.0001↓	<0.0001↓	<0.0001↓	<0.0001↓	—	—
H2O2 production
Compared with control	0.0005↓	NS	0.0013↓	NS	0.0003↓	NS	0.003↓	NS	0.0006↑	0.0002↑
Compared with BAC	<0.0001↓	<0.0001↓	<0.0001↓	<0.0001↓	<0.0001↓	<0.0001↓	<0.0001↓	<0.0001↓	—	—

 NS, nonsignificant; ↓, decrease in fluorescence; ↑, increase in fluorescence.

*  Concentration (%).

Figure 2.

 

Cellular viability (neutral red test), DNA condensation (Hoechst 33342 test), O₂ ^⨪ production (HE test), and H₂O₂ production (dichlorofluorescein diacetate [DCFH-DA] test) evaluations after 15 minutes of treatment and 24 hours of recovery with carbomer 934P, hyaluronic acid, or BAC. Results are expressed as a percentages of control values (solid horizontal line). (A) Hydrogels tested at their usual concentrations (unpreserved and preserved HA, 0.18%; unpreserved and preserved carbomer, 0.3%) and BAC tested at 0.005%. (B) Hydrogels and BAC tested after a 1:10 dilution in normal culture medium. *P < 0.05; **P < 0.01, and ***P <0.001 compared with the control.

Table 2.

 

Statistical Analysis Summary after 15 Minutes of Treatment Followed by 24 Hours of Cell Recovery

	Carbomer 934P/BAC−			Carbomer 934P/BAC+			Hyaluronic acid/BAC−			Hyaluronic acid/BAC+			BAC
		0.3	0.03	0.3	0.03	0.18*	0.018	0.18	0.018	5 × 10−3	5 × 10−4
Cellular viability
Compared with control	0.0034↓	NS	0.002↓	NS	NS	NS	NS	NS	0.0004↓	0.04↓
Compared with BAC	<0.0001↑	<0.05↑	<0.0001↑	<0.05↑	<0.0001↑	NS	<0.0001↑	<0.05↑	—	—
DNA condensation
Compared with control	0.0235↓	NS	0.013↑	NS	NS	NS	NS	NS	<0.0001↑	0.0011↑
Compared with BAC	<0.0001↓	<0.0001↓	<0.0001↓	<0.0001↓	<0.0001↓	<0.0001↓	<0.0001↓	<0.0001↓	—	—

 NS, nonsignificant; ↓, decrease of fluorescence; ↑, increase of fluorescence.

*  Concentration (%).

Figure 3.

 

F-actin detection using phalloidin: (A) control cells and cells treated with (B) BAC 0.005%, (C) hyaluronic acid 0.18%, and (D) hyaluronic acid 0.18% associated with BAC 0.005%. A decrease in cell size associated with cell disorganization and chromatin condensation was observed with BAC, whereas unpreserved and preserved hydrogels did not induce any morphologic changes. Magnification, ×1000.