1. Introduction
There are several approaches to soft tissue reconstruction of the oral cavity focused on restoring aesthetic and natural features [1] . Such procedures are frequently required to promote proper healing following deficiencies caused by tumor excision, fissures, trauma, dental implants, and gingival recessions [2] .
Autogenous grafts remain the gold standard for cases that require gingival augmentation surgery, with unmatched success rates [3] . Soft tissue grafts may be harvested from different donor sites, such as the palate, the retromolar region and the edentulous spaces [4] [5] [6] . The disadvantages of harvesting the graft from the retromolar pad and edentulous sites are a limited amount of tissue availability and the recovery of only thinner grafts. As a result, the palate is the preferred site for soft tissue graft harvesting [7] , which demands a second surgical site, increasing morbidity in terms of post-operative discomfort and procedure time [5] . Therefore, various options should be considered in order to avoid a second surgical site while also reducing postoperative time and complications, potentially increasing patient acceptance of this type of procedure [8] .
Three-dimensional porcine collagen matrix has been introduced as soft tissue graft substitutes in periodontal plastic surgery [8] . Collagen is an important biomaterial for medical applications due to its biological characteristics such as bio and cytocompatibility and biodegradability [9] . Additionally, its hemostatic function enables early wound stabilization, fibroblast attraction and semi-permeability, promoting nutrient exchange [10] .
Most matrices are primarily composed of type I and III collagens derived from pigs, cattle, and humans [11] , which are commonly used in membrane production [12] . Type I collagen is found in the majority of connective tissues in the body [13] , while type III collagen is found primarily in the skin and vessels [14] . Although different in terms of the three-dimensional arrangement of the molecules, both share the same molecular structure, and act together in the formation of fibrillar aggregates [14] .
Two of the collagen membranes currently available as soft tissue substitutes are Fibro-Gide® (Geistlich Biomaterials) and Mucoderm® (Botiss Biomaterials). Both are made of porcine collagen and are indicated to replace the subepithelial connective graft. Besides types I and III collagens, Mucoderm® contains elastin, and Fibro-Gide® is a porous, resorbable matrix that is chemically cross-linked to preserve its volume stability.
Although clinical studies have demonstrated excellent tissue regeneration with both matrices, additional studies that report on cell viability, and the potential to induce connective tissue synthesis are, however, lacking. Therefore, the aim of this study was to assess the biological behavior of both matrices on human gingival fibroblasts, as well as their ability to induce the production of type I collagen, metalloprotease-2 and inhibitors (TIMP-1 and TIMP-2), which are important in the early stages of oral cavity soft tissue formation.
2. Materials and Methods
2.1. Sample Groups
Two resorbable collagen matrices of animal origin (porcine) were used, Fibro-Gide® (Geistlich Biomaterials) and Mucoderm® (Botiss Biomaterials), at 30 × 40 mm. Square samples at 5 × 5 mm were obtained from the matrices and human gingival fibroblast cells obtained from three different donors were plated on the membranes, after approval by the Ethics Committee of the São Leopoldo Mandic Research, Campinas, Brazil (#3,833,067). The cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) (Nuticell®, Campinas SP, Brazil) supplemented with 10% fetal bovine serum (Cultilab®, Campinas SP, Brazil) and 1% antibiotic-antimycotic solution (penicillin-streptomycin) (Sigma, St. Louis, Missouri, USA). This procedure was performed in each membrane separately. The 96-well plates were kept in an incubator at 37˚C.
2.2. Topographic Analysis of the Biomaterials
The morphology and average roughness (Ra) of the different surfaces were evaluated. The ultrastructural morphology of the samples was evaluated using a high-resolution scanning electron microscope (Quanta FEG 250; FEI Company, Eindhoven, The Netherlands). Three specimens of each surface were mounted directly on stubs and photographed at 1000× and 20,000× magnification.
For the analysis of the average surface roughness (Ra), 4 fragments of each surface were measured with a tip instrument (profilometer, Mitutoyo Surftest SJ-200, Japan). Four linear measurements were conducted on each sample according to DIN ISO 1302 standards and the arithmetic mean of the surface roughness profile (Ra) was calculated for each sample.
2.3. Cell Viability Assay
Cell viability of the cell culture plated on the different membrane surfaces was measured using MTT assay. After 24, 48 and 72 h of cell plating, 10 µL of MTT solution (5 mg/mL, Sigma, USA) diluted in DMEM serum-free medium was added to the treated cultures, which were then incubated for 3 hours at 37˚C. After the incubation, 100 µL of 10% DMSO solution (Dimethylsulfoxide, LGC, São Paulo, Brazil) was added.
After the crystals were solubilized, an ELX800 microplate reader (Epoch Biotek Instruments, Inc.) at 590 nm was used for quantification, and the optical density (OD) measures were obtained.
2.4. Enzyme Immunoassay of Collagen Type I, MMP-2 and TIMP-1 and TIMP-2 Inhibitors (ELISA)
After 24, 48 and 72 h, the supernatants from the cell cultures were harvested and centrifuged at 5000 g for 15 min at 4˚C. Aliquots of each sample were assayed by means of ELISA to determine the type I collagen, MMP-2 and inhibitors (TIMP-1 and TIMP-2) levels, according to the manufacturer’s recommendations (R&D Systems, USA). Total type I collagen, TIMP-1 and MMP-2 were quantified in picograms per mL (pg/mL). The results were calculated using the standard curves created in each assay. The ELISA assays were performed in a blind fashion in triplicate.
2.5. Statistical Analysis
Descriptive and exploratory analyses of all data were performed. They indicated that the data did not meet the assumptions of an analysis of variance (ANOVA). Generalized linear models (GLMs) considering main effects and the interaction between them were then used. Analyses were conducted using the R program with a significance level of 5%.
3. Results
3.1. Surface Topography
The ultrastrucutral morphology of the surfaces is represented in Figure 1 by scanning electron micrographs. Both matrices exhibited a porous structure on the surface, enabling the ingrowth of cells after grafting. Mucoderm® presented collagen fibers set in a loose mesh-like arrangement, while Fibro-Gide® exhibited a sheet-like amorphous matrix.
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Figure 1. Representative SEM images of Fibro-Gide® ((A), (B)) and Mucoderm® ((C), (D)) membranes. Caption: Bars: (A) = 500 µm, (B) = 50 µm, (C) = 10 µm, (D) = 5 µm.
Regardless of the surface pattern, the roughness mean values for the Mucoderm® were statistically higher (7.87 ± 0.83 µm) than for Fibro-Gide® (6.04 ± 0.36 µm) (Figure 2).
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Figure 2. Average surface roughness (standard deviation), in µm, and interferometry of the surfaces of Fibro-Gide® (A) and Mucoderm® (B) membranes. Caption: *representation of the statistical difference between groups (p < 0.05).
3.2. Cell Viability
Cell viability at the different analysis times for all groups is shown in Table 1. The results of the MTT assay revealed that there was no difference in the viability of cells grown on Fibro-Gide® and Mucoderm® membranes compared with polystyrene (control, C), for each time point (p > 0.05). Additionally, no difference was observed regarding the viability of cells for each group at the different times evaluated (p > 0.05).
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Table 1. Cell viability assay at the different analysis times.
Caption: Same letters (upper case horizontally and lower case vertically) indicate that there were no statistically significant differences (p > 0.05).
3.3. Quantification of Type I Collagen, MMP-2 and TIMP-1 and TIMP-2 Inhibitors
The quantification of the proteins secreted by the fibroblasts plated on the different membranes is shown in Table 2. Lower levels for type I collagen, MMP-2 and TIMP-1 were observed for the cells plated on Mucoderm® than on the C and Fibro-Gide®, at all experimental points evaluated (p < 0.05). In relation to TIMP-2, after 24 and 48 h, the secretion levels were lower on Mucoderm® than on Fibro-Gide® and C (p < 0.05). After 72 h, no statistical differences were observed (p > 0.05).
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Table 2. Quantification of collagen I, MMP-2, TIMP-1 and TIMP-2 secreted by fibroblasts plated on the different surfaces. Mean values (standard deviation) expressed in pg/mL.
Caption: Distinct letters (upper case horizontally and lower case vertically) indicate statistically significant differences (p < 0.05).
4. Discussion
Periodontal plastic surgery is often performed to correct defects of the gingiva, alveolar mucosa and bone. For this purpose, the harvest of autogenous connective tissue graft is a well established treatment. However, autogenous grafting techniques are associated with some level of morbidity, particularly if the donor site is limited. To overcome this issue, new treatment options are being developed, using substitute biomaterials (e.g. xenografts), in order to reduce the number of surgeries as well as intraoral donor sites [15] .
The use of xenogenous collagen matrices has increased lately, since they can effectively promote root coverage and tissue thickness gain, with successful rates compared to autogenous connective tissue grafts [15] . Moreover, such substitutes can reduce the morbidity, avoiding the second surgical site (i.e. donor area) [16] [17] . Recent findings have shown that collagen matrices are equally effective for both increasing thickness and increasing the band of keratinized mucosa around implants [18] [19] .
Mucoderm® and Fibro-Gide® are porous resorbable matrices of porcine dermis origin used as a substitute in cases of loss of the connective tissue structure. These matrices are composed of type I and III collagen with a three-dimensional structure similar to human connective tissue. According to the manufacturer, Mucoderm® is a resorbable matrix of 1.2 to 1.7 mm thickness, with no artificial or chemical cross-linking, constituted by 60% - 96% (w/w) porcine collagen and 4% - 40% (w/w) elastin. On the other hand, Fibro-Gide® is constituted only by collagen exposed to chemical cross-linking to preserve its volume [20] . The surface topographies of both matrices evidenced the roughness and open-porous structure which enables the ingrowth of soft tissue cells and blood vessels. In the present study, it was verified that Mucoderm® presented collagen fibers set in a loose mesh-like arrangement, while Fibro-Gide® exhibited a sheet-like amorphous matrix. Despite the similarity concerning the porous structure, the roughness average of Mucoderm® was slightly higher than that of Fibro-Gide® (p < 0.05).
The most favorable conditions for efficient cell adhesion, growth, and proliferation are dependent on cell type. Roughness average higher than 2 µm stimulates the formation of focal adhesion points, which are required for healthy cellular functioning. This context allows a maximum cell-substrate interaction resulting in stable adhesion, and, as a result, the highest cell growth and proliferation [21] . Regardless of the slight differences between both matrices, they are considered to have high roughness, and the viability cell assay revealed no difference between the matrices and the control group. However, concerning the production of extracellular matrix proteins, the results of the present study revealed that fibroblasts plated on the FibroGide® secreted more type I collagen, MMP-2, TIMP-1 and TIMP-2 than Mucoderm®.
Fibro-Gide® is made of reconstituted collagen and receives a cross-linking which prolongs the enzymatic degradation of the product [22] , enhancing the subsequent cell repopulation [23] [24] . Despite structural advantages, artificial cross-linked treatment negatively interferes with biocompatibility and may hinder tissue integration as well as vascularization, potentially contributing to increase wound dehiscence and oral exposure [25] [26] [27] . Although the scientific literature suggests that collagen cross-linking has biological disadvantages, some studies suggest that, when compared to matrices without cross-linking, this treatment balances mechanical volume stability with cell compatibility, contributing to cell growth and vascularization [7] [28] . In fact, this data supports the findings of the present study with higher type I collagen, MMP-2, TIMP-2 and TIMP-2 synthesis when cells were plated on Fibrogide® matrix than on Mucoderm®.
Type I collagen is the main protein of the connective extracellular matrix that provides structural support for a variety of tissues, allowing cell adhesion and migration during wound healing [29] . This may support the clinical studies that demonstrate the regenerative capacity of the matrix in replacing autogenous connective tissue grafts [30] [31] .
MMP-2 belongs to the family of matrix metalloproteinases, a group of endopeptidases that participate in the degradation of several extracellular matrix proteins [32] , playing an important role in remodeling, osteogenesis, and healing. MMP-2 deregulation can participate in the progression of a variety of physiological and pathological functions such as tissue repair and inflammation [33] . TIMPs are specific endogenous inhibitors of MMP pro-enzyme activity [34] and can inhibit different MMP with varying degrees of efficacy. The MMP/TIMP ratio can influence the specific activities of the metalloproteinases. Furthermore, during inflammation, the expression or activity of MMPs, which are secreted by proinflammatory cells, increases [32] .
The results of the study indicate an increase in MMP-2 and TIMP-1 and TIMP-2 on cells plated on Fibro-Gide®, possibly indicating a greater ability of this matrix to stimulate periodontal and peri-implant tissue remodeling when compared with Mucoderm®. However, it’s important to state that Mucoderm® can be considered an alternative to autogenous connective tissue graft for root coverage purposes [35] . Further in vivo studies using both matrices are required to determine whether the Fibro-Gide® matrix is in fact capable of promoting tissue remodeling in a beneficial way in terms of clinical results, or whether it induces more intense inflammation during healing.
5. Conclusion
In conclusion, the results indicated that, despite the fact that both matrices demonstrated cell viability, Fibro-Gide® induced an increase in type I collagen, MMP-2 and TIMP-1 and TIMP-2, which might contribute to periodontal tissue remodeling when compared to Mucoderm®.
Acknowledgements
The authors would like to thank Marília Portela for promptly volunteering to review this manuscript regarding its English language content and Dr. Rafael Bovi Ambrosano for helping with the statistical analysis of the data.
Funding
This work was supported by Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)—Research Productivity Fellowship to Elizabeth Ferreira Martinez (#404935/2021-4).