Comparison of Attachment and Proliferation of Human Gingival Fibroblasts on Different Collagen Membranes.

BACKGROUND AND AIM: Human gingival fibroblasts cultured on collagen membrane as an alternative treatment method used in tissue regeneration can lead to improved results in root coverage. The aim of this study was to evaluate the human gingival fibroblast proliferation and adhesion cultured on three types of collagen membranes.
MATERIALS AND METHODS: In this in vitro study, first-line human gingival fibroblast cells (HGF1-RT1) prepared and cultured on three membranes, including porcine pericardium (PP) (Jason, Botiss dental), human pericardium (HP) (Regen, Faravardeh Baft Iranian), and glutaraldehyde cross-linked (GC) (BioMend Extend, Zimmer Dental). Cell survival was assessed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) after 24, 48, and 72 h and 7 days. Furthermore, morphology and adhesion of cells on the membrane were evaluated after 1 and 7 days by electron microscopy (scanning electron microscopy [SEM]). Statistical analysis was performed using two-way ANOVA with a significance level of 0.05.
RESULTS: Based on the results of MTT, cell survival on HP and PP membranes after 7 days significantly increased (P < 0.001), but for the GC membrane, it was reduced after 7 days (P = 0.031). Cell survival on HP and PP membranes did not differ (P = 1) and was more than GC (P < 0.001). SEM images showed that the adhesion of cells was better on HP and PP membranes than GC.
CONCLUSION: The results of this study showed that natural collagen membranes (HP and PP) similarly support proliferation and adhesion of gingival fibroblasts. Survival and adhesion of gingival fibroblasts on cross-linked collagen membrane was less than two other membranes.

INTRODUCTION

Gingival recession is a common clinical problem that could cause tooth hypersensitivity, pain, root caries, and esthetic issues.[1] The routine treatment modality is soft-tissue autograft which is associated with donor-site morbidity and patient discomfort. Application of tissue engineering by culturing fibroblasts on membranes, however, does not require donor site while providing unlimited amount of graft in a relatively shorter treatment time.[2] In addition, following guided tissue regeneration (GTR) in periodontal surgery, in which membranes are necessary,[3] it has been demonstrated that regeneration of fibers and cementum as well as limited new bone formation could be expected.[45] Furthermore, for root coverage, GTR has been associated with less failure compared to coronally advanced flap surgery.[6]

Pitaru et al.[789] and Blumenthal et al.[10111213] showed that collagen membrane could be used for GTR. It has been demonstrated that the results of application of collagen membranes in GTR are comparable to those of expanded polytetrafluoroethylene membranes.[3141516]

Fibroblasts are the most common gingival cells and have a vital role in soft-tissue regeneration. Using a proper scaffold, which supports adhesion and proliferation of fibroblasts, they could accelerate tissue repair. In this regard, some of the current tissue engineering approaches involve ex vivo fibroblast expansion on membranes followed by their implantation at the defect.[17181920] Collagen membrane with autologous gingival fibroblast has been introduced as a contemporary approach in GTR.[20]

Collagen membranes could be used either native or processed. Although there are several covalent bonds in natural collagen fibers, they could be processed to increase their strength. One of the most common chemical procedures on collagen membranes is cross-linking which could be done physically or chemically and would increase covalent bonds and strength of collagen fibers.[82122] However, the effect of cross-linking on bioactivity and biocompatibility of collagen membranes has been less issued. Hence, the aim of the current in vitro study was to compare the human fibroblast adhesion and proliferation on two native collagen membranes compared to a cross-linked one.

MATERIALS AND METHODS

Membranes

In this in vitro study, two native collagen membranes including human pericardium (HP) membrane (Regen, Faravardeh Baft Iranian, Tehran, Iran) and porcine pericardium (PP) membrane (Jason membrane, botiss dental GmbH, Berlin, Germany) and one glutaraldehyde cross-linked (GC) collagen membrane (BioMend Extend, Zimmer Dental, Carlsbad, CA, USA) were used. HP is a 0.6–1.2-mm thick allograft membrane which includes basement membrane on one side. PP is a relatively thin (0.1–0.2 mm) membrane with high amount of collagen type III to increase its strength and resorption time which is designed for oral surgeries.[23] GC, on the other hand, is a chemically cross-linked bovine collagen membrane with a mean 0.4-mm thickness.[24]

Human fibroblast cell culture

Human gingival fibroblast cells (HGF1-RT1, Pasteur, Tehran, Iran) were cultured in DMEM (Gibco Laboratories, Grand Island, NY, USA) culture medium supplemented with 10,000 IU/ml penicillin (Gibco), 100 mg/ml streptomycin (Gibco), and 10% FBS (Gibco) in 37°C, 98% humidity, and 5% CO2. Culture media were changed three times a week until cells reached subconfluent stage. Then, they were removed using 0.25% trypsin/EDTA and passaged. The fourth passage cells were used in the following experiments.

Cell seeding

Collagen membranes were cut into 5 mm × 5 mm pieces under sterile condition and rinsed three times with sterile saline for 10 min. They were placed in 96-well plates and each one was seeded with 6 × 104 human gingival fibroblasts. According to previous in vitro studies,[2526] six samples for each group at each time point were used. No membrane was used in the control group and cells were seeded in empty wells. Following cell adhesion at 37°C for 1 h, the plates were incubated at 37°C in a humidified atmosphere containing 5% CO2.

3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide

To assess the amount of vital cells in each group, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) test was performed at 24, 48, and 72 h and on 7 days after cell seeding using MTT (Sigma, USA) at described elsewhere.[27] The absorbance was determined by a microplate reader (Anthos, Austria) at 590 nm wavelength. Proliferation was estimated by comparing the MTT values at different time points.

Scanning electron microscopy

To visually observe cell attachment, scanning electron microscopic (SEM) images were taken 1 and 7 days after cell seeding. First, cells were fixed by 2.5% glutaraldehyde for 2 h and 1% osmium for 1 h followed by dehydration using sequential concentrations of ethanol. Then, they were sputtered with gold and observed under an SEM (VEGA, TESCAN, Czech Republic) at 10 kV.

Statistical analysis

A two-way ANOVA test followed by Tukey post hoc test was applied for the comparison of MTT values among different groups at different time points. Statistical analysis was performed using computer software (SPSS, Version 18, SPSS Inc., Chicago, IL, USA), with a significance level of 0.05.

RESULTS

The trend of cell adhesion (MTT values) on different membranes is demonstrated in Figure 1. As it is apparent, the number of vital cells on all groups except GC was increased over time. Two-way ANOVA showed that the difference between groups and time points was statistically significant (P < 0.001) [Table 1].

Figure 1

Comparison of the mean 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide values in different collagen membrane compared to control group at different time points

Table 1

Two-way ANOVA test for comparison of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide values between different groups at different time points

A pairwise comparison of study groups revealed that control group had the most and GC membrane had the least amount of cells (P < 0.001) while the difference between HP and PP membranes was not statistically significant (P = 1) [Table 2].

Table 2

Pairwise comparison of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide values between collagen membranes and control group

Furthermore, comparison of different time points showed that only 7-day cultures had significantly more viable cells (P < 0.001) and the difference between shorter culture times was not significant (P > 0.05) [Table 3].

Table 3

Pairwise comparison of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide values between study time points

SEM images of membraned cultured with fibroblasts for 24 h show that fibroblasts were attached on the surface of all the study membranes [Figure 2a–f]. However, after 7-day culture, no cell was observed on GC membrane while fibroblasts were still attached on HP and PP native collagen membranes [Figure 3a–f].

Figure 2

Attachment and distribution of human gingival fibroblasts after 24 h culture on (a and b) glutaraldehyde cross-linked, (c and d) porcine pericardium, and (e and f) human pericardium collagen membranes

Figure 3

Attachment and distribution of human gingival fibroblasts after 7 days culture on (a and b) glutaraldehyde cross-linked, (c and d) porcine pericardium, and (e and f) human pericardium collagen membranes

DISCUSSION

Membranes could prevent tissue ingrowth toward periodontal defects and clot stabilization as well as preserve new formed bone. They also could increase the concentration of growth factors and osteogenic cells.[28] In this study, biocompatibility of three collagen membranes and the response of human gingival fibroblasts toward them were investigated. The results showed better response of native collagen membranes compared to cross-linked one.

The results showed that the number of attached fibroblasts on HP and PP native noncross-linked collagen membranes was increased from 1 to 7 days’ culture. However, cell population was decreased on GC collagen membrane during 7-day culture. Attachment of surface-dependent cells is essential for cell proliferation.[29] The results of SEM which was used for the observation of cell attachment are also in agreement with MTT test. SEM images showed that fibroblasts were attached to all three membranes, while after 7 days, no cell was found on GC membrane.

The chemical method which is used for cross-linking in the BioMend extend membrane is through application of glutaraldehyde. Although this method is a common approach, there are some reports on cytotoxic effect of the remnant aldehydes.[3031] Some have suggested that use of glutaraldehyde does not have a significant influence on regenerative treatments using GC membranes.[32] However, the results of the current study showed that cell adhesion on GC membrane decreased during 7 days which might be due to gradual release of aldehydes in the culture media. Toxicity of glutaraldehyde is dose dependent[33] and its aggregation during 7 days might be the reason for reduction of cell population on GC membrane. The mean MTT values for GC after 7 days were about 5% of the control group. This might show that GC membrane is not compatible with human gingival fibroblasts. Similarly, it has been demonstrated that GC membrane could decrease its biocompatibility to a toxic level.[303134] Speer et al.[30] reported that products released from GC membranes have a cytotoxic effect. However, van Luyn et al.[35] showed that constant change of cell substrate would prohibit toxic effect of these membranes on fibroblast cells. Changing cell media was done to mimic wash-out effect of body fluids. Nonetheless, implantation of GC membranes in vivo resulted in foreign body and inflammatory reactions in 10 days.[36] It seems that expected removal of toxic products needs more time and primary inflammation is inevitable.

To the extent of authors’ knowledge, no previous study compared membranes used in the current experiment. The results of the current study is in agreement with some of previous studies which reported that noncross-linked membranes support cell adhesion and proliferation.[373839] It has been reported that PP collagen membrane (Jason membrane) can support proliferation SaOS-2 osteoblasts during 7-day culture.[23] Further, SEM observation of this membrane revealed that it has interconnected pores which would permit cell infiltration[23] and its surface topography supports cell adhesion.[40]

Two studies reported that cross-linking does not necessarily cause a reduction in cell proliferation.[4142] The methods for cross-linking in these studies were by nordihydroguaiaretic acid and chitosan. However, the results of the current study revealed that cross-linking by glutaraldehyde would cause a significant reduction in the proliferation of human gingival fibroblasts. This reduction might be due to the effect of production method rather than cross-linkage.

Clinical significance

On the one hand, membranes used in GTR procedures should be obviously compatible to the cells including gingival fibroblasts. On the other hand, when primary wound closure is not achievable during periodontal surgery, cell migration and attachment are necessary for closure and preventing infection.[43] Fibroblasts are among the most important cells that should attach properly to the exposed membraned.[44]

Furthermore, as the fibroblast can produce collagen and growth factors, their application in regenerative medicine in the field of periodontal surgeries has increased.[2045] The common approach is ex vivo proliferation of fibroblasts followed by their transplantation to the defect site using proper scaffold.[1946] Various materials including collagen membranes have been used as scaffold.[1945] The results of this study showed the ability of two native collagen membranes for fibroblast support.

However, it should be considered that the current study was an in vitro experiment and the effect of several confounding factors such as body fluids, recipient site, and cell interactions was not assessed. Therefore, extrapolation of the results to the clinical situations should be done with precaution. In vitro studies, nonetheless, allow controlling all the factors and evaluation of the aimed variable which is not easily achievable in in vivo studies. Another limitation of this study was that we did not measure release of glutaraldehyde or other products from studied membranes. Further studies evaluating the amount of toxic products released from membranes could reveal the causal relationship between toxicity and glutaraldehyde released from GC membrane.

CONCLUSION

Considering the limitations of the current in vitro study, the results could be summarized as follows:

Viable human gingival fibroblasts were increased on PP and HP natural collagen membranes from 24 h to 7 days culture while it was decreased on the cross-linked collagen membrane

These was no significant difference between amount of living cells on HP and PP membranes and both were significantly more than GC membrane

Morphology and attachment of cells on three membranes were relatively similar after 24-h culture while no cell was observed on GC membrane after 7-day culture.

These results show that natural collagen membranes support attachment and proliferation of the human gingival fibroblasts while GC collagen membrane does not. In addition, the results showed the toxic effect of GC membrane on the human gingival fibroblasts.

Financial support and sponsorship

This study was funded by the School of Dentistry, Shahid Beheshti University of Medical Sciences.

Conflicts of interest

There are no conflicts of interest.