|Year : 2020 | Volume
| Issue : 5 | Page : 763-767
|Development of a membrane for guided tissue regeneration: An in vitro study
Ramon C Fernandes1, Mário Igor Damasceno1, Gabriel Pimentel1, Juliano S Mendonça2, Maria V Gelfuso3, Sérgio L da Silva Pereira4, Vanara F Passos2
1 Doctor of Dental Surgery, School of Dentistry, University of Fortaleza, Fortaleza, Ceará, Brazil
2 Federal University of Ceara, Faculty of Pharmacy, Dentistry and Nursing, Fortaleza, Ceará, Brazil
3 Federal University of Itajubá, Institute of Mechanical Engineering, Itajubá, Minas Gerais, Brazil
4 University of Fortaleza, School of Dentistry, Fortaleza, Ceará, Brazil
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|Date of Submission||12-Mar-2019|
|Date of Decision||05-Sep-2019|
|Date of Acceptance||06-Apr-2020|
|Date of Web Publication||08-Jan-2021|
| Abstract|| |
Aim: The aim of this study was to develop an alternative low-cost membrane for use in guided tissue regeneration (GTR). Setting and Design: In vitro study. Methods and Material: In this study, a membrane prepared from a 335 mm sized opening nylon substrate, covered in aqueous resin derived from chitosan, was compared with a commercial material, a non-degradable expanded poly (tetrafluoroethylene). Nylon substrate samples 2.0 × 2.0 cm were covered by aqueous resin based on diluted chitosan solution into 1:05 or 1:10 by spin coating technique to produce from 06, 10, and 15 layers. The surfaces of these membranes were observed using optical microscopy. The physical properties were measured by hydration superficial energy measurements (ΔG) and a tensile test machine. Statistical Analysis: Statistical analysis was performed using the Student's t test at a significance level of 5%, using the BioEstat 2.0 program. Results: The Δ G values of the nylon membrane covered by the 1:05 of chitosan with 15 layers were close to the commercial membrane's Δ G values. The tensile strength values of the nylon membrane covered by the 1:05 of chitosan with 15 layers were higher than the commercial membrane's (115.826 MPa, P < 0.05). Conclusion: Therefore, the membrane developed shows some favorable physical properties that could qualify it as a material candidate for use in guided tissue regeneration.
Keywords: Chitosan, guided tissue regeneration, membranes, nylon
|How to cite this article:|
Fernandes RC, Damasceno MI, Pimentel G, Mendonça JS, Gelfuso MV, da Silva Pereira SL, Passos VF. Development of a membrane for guided tissue regeneration: An in vitro study. Indian J Dent Res 2020;31:763-7
|How to cite this URL:|
Fernandes RC, Damasceno MI, Pimentel G, Mendonça JS, Gelfuso MV, da Silva Pereira SL, Passos VF. Development of a membrane for guided tissue regeneration: An in vitro study. Indian J Dent Res [serial online] 2020 [cited 2021 Jan 28];31:763-7. Available from: https://www.ijdr.in/text.asp?2020/31/5/763/306444
| Introduction|| |
Periodontitis is a problem that causes the loss of periodontal tissues and, if untreated, it will generate serious problems, such as mobility and dental movement, pain, and tooth loss. López et al. verified an improvement of periodontal health in the last decades, but the prevalence remains elevated. The ideal goal of periodontal treatment is the regeneration of the periodontal tissues lost after the evolution of periodontitis., Guided tissue regeneration (GTR) is the technique that use membranes made of nonresorbable and resorbable materials, such as polytetrafluoroethylene and collagen, so it is biocompatible, safe, nonallergic, nontoxic, and have no risk of disease transmission.,,
To regenerate a tissue, a structure that acts as a temporary matrix for cell proliferation and deposition of extracellular matrix is required. Therefore, biomaterials are used to provide support and framework for cell growth. Nylon, a biocompatible material, is a long chain polymer that is available in monofilament and multifilamentar forms. Its chain is symmetric and has NH and C = O regularly spaced throughout. They attract each other through hydrogen bonds, resulting in a high-mechanical strength (tensile strength and flexibility). In addition to the mechanical strength, nylon has a high endurance and chemical resistance. In addition, this material presents easy handling and sterilization, has a low cost, and it is well tolerated by the organism.,
The introduction of nanotechnology has improved the properties of various types of fibers. In this field, biomaterials have garnered substantial interest in tissue engineering. Biomaterials can be synthetics (e.g. poly (ethylene glycol), poly (lactic-co-glycolicacid), poly (ethylene terephthalate), and polygly-colic acid) or naturals (e.g. naturally occurring polymers such as chitosan, purified extracellular matrix, and extracellular matrix derived by decellularization).
The chitosan appears to be a feasible alternative to accomplish the tissue engineering because of its biological features, ability to be formed into various configurations, and capacity to sustenance osteoblast attachment., Chitosan is a hydrophilic biopolymer obtained from chitin. Its primary natural source is the crustacean carapace, with a variety of applications mainly in the textile, food, and cosmetics industries., The chitosan-based biomaterials are being tested in the treatment of periodontal bone defects. Some clinical studies that test its use do not verify inflammatory or allergic problems after its topical application, consumption, implantation or injection into the human body.,,, In addition, it shows to have a suitable degradation rate and ability to inhibit growth of different bacteria.,, Chitosan's chemical structure, similar to that of hyaluronic acid, strengthening this indication for use as a repairing and therapeutic agent.,
Although some reports show studies using chitosan as a biomaterial, research has failed to characterize the chitosan tested, not relating physical factors such as superficial hydration energy and mechanical characterizations and the influence of these features in the results obtained, thus opening more fields for studies regarding its properties. Therefore, in vitro studies focusing on all these parameters are still needed. Thus, the goal of this study is to evaluate the physical properties of a new chitosan/nylon membrane for a potential use in the GTR.
| Material and Methods|| |
Production of chitosan/nylon membrane
The production of the membrane was started with beads of chitosan found commercially (Sigma-Aldrich >98%, St. Louis, MO, USA). These granules were dissolved in a beaker containing distilled water and acetic acid for 24 h. Thereafter, the solution was diluted with distilled water in different ratios to produce resins with several viscosities. The mixtures were stirred under heating at 50°C using a magnetic stirrer in order to completely dissolve the chitosan beads, forming a smooth and homogenous resin.
TYLER nylon meshes (opening of 335 μm; São Paulo, Brazil) were obtained commercially and washed with soap and water, then dried in air. They were cut into 2.0 × 2.0 cm2. This mesh was chosen because it is easily found in local stores at low costs. The meshes were fixed in the equipment used to deposition of chitosan by rotation. This equipment was built at the Laboratory of Materials and Electronic Instrumentation-LaMatIE-UNIFOR, but similar equipment can also be purchased commercially. It is used to rotate a substrate at different speeds when one or a few drops of a solution touches the rotating substrate. The liquid is spread, forming a homogeneous thin film.
Initially, resins with different dilutions (1:5 and 1:10) were made with 6, 10, or 15 layers. The layers were built using the substrate rotation at 10,000 rpm for 15 s, and subsequently dried using a 250 W in candescent lamp at a distance of 15 cm for 6 min. After drying, the membrane was taken to an autoclave for complete sterilization. In comparison to this novel membrane, a commercial nondegradable, expanded polymaterial (tetrafluoroethyleneECOFLON; Rio de Janeiro, Brazil) was tested.
The chitosan/nylon membranes were examined using an OLYMPUS microscope (Center Valley, PA, USA) (×400, ×2000, and × 4000 magnification) to evaluate the quality of coverage of the resin on the nylon mesh and the final microstructural homogeneity. From these observations, it was decided to prepare the membranes obtained from the deposition of resins with dilutions of 1:05 and 1:10. For each dilution, membranes with 6, 10, or 15 layers were selected for the tests that were conducted to determine hydration energy for the membranes.
Superficial hydration energy characterization
This test consisted of depositing a drop of 15 μl deionized water on the membrane. Adjusting the position of a digital camera (Olympus DP72), the left drop over the membrane was imaged so that the angle of view permitted the view of the thickness of the membrane as a line, as illustrated in [Figure 1]. This test was also conducted for the pure nylon mesh washed and dried in air, as well as a commercial brand membrane [Figure 1]. Subsequently, wetting angles were measured using Gimp software. The YoungDupré equation [−ΔG=(1 + cos θ)γ] was used to calculated the hydration energies of the membranes, ΔG, where θ is the contact angle between water and the membrane surface and γ is the water tension surface (73 mJ/m2). There were no measurements for the pure nylon, which is the mesh without resinous deposition, because it showed no reproducibility during the test due to its permeability and easy wetting.
|Figure 1: Drop of water on a (a) Commercial membrane and (b) 15 layer resin membrane 1:05, illustrating the test for determining the hydration energy of membranes|
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Uniaxial tensile tests on the 1:05 chitosan/nylon membrane (15 layers), nylon membrane, and commercial product were performed on a universal tensile testing machine, model INSTRON 4484 (São José dos Pinhais, PR, Brazil), applying 15 tons at 5 mm/min. This membrane was chosen due to the observed results of its hydratation energy. The tensile strength on maximum load (MPa) and elongation at maximum load were determined. Five samples were tested for each composition.
| Results|| |
The mean values and standard deviations were calculated for each group. Statistical analysis was performed using the Student t test at a significance level of 5%, using the BioEstat 2.0 program.
[Figure 2] shows the microstructural surfaces aspects of the (a) commercial membrane (ECOFLON membrane), (b) 6 layers of resin membranes 1:05, and (c) 6 layers of resin membranes 1:10. It can be seen that the ECOFLON membrane [Figure 2]a shows no permeability, even with the maximum possible optical magnification (×2000). [Figure 2]b and [Figure 2]c illustrates the insufficient coverage of the resin in both dilutions of the 6 layers of chitosan.
|Figure 2: Microstructural surfaces aspects of the (a) Commercial membrane (ECOFLON membrane), (b) 6 layer resin membrane 1:05, and (c) 6 layer resin membrane 1:10. Optical magnification × 2000|
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The membranes with a larger number of layers, i.e., with 10 and 15 layers, correspond to the layers 1:0510 [Figure 3]a and layers 1:0515 samples [Figure 3]b, respectively. It is observed a better coverage and closing the holes, in particular it may be noted that there is a higher quality coverage for the sample layers 1:0515.
|Figure 3: Surfaces aspects of (a) 10 layer resin membrane 1:05 and (b) 15 layer resin membrane 1:05. Optical magnification × 400|
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At a higher magnification, there is a pattern of light diffraction in [Figure 4]a. The film formed in the holes of the nylon mesh has the characteristic of being formed thinner than those for the samples of 15 layers of resin membranes 1:10 [Figure 4]b. The quality of the coverage of the nylon mesh film is essential to set the hydration energies of the membranes.
|Figure 4: Microstructural surfaces aspects of (a) 15 layer resin membrane 1:05 and (b) 15 layer resin membrane 1:10. Optical magnification × 4000|
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[Table 1] shows the values of the hydration energies (ΔG) of the commercial membrane and 10 layers of resin membranes 1:05 samples. The values of hydration energies (ΔG) for the 15 layers of resin membranes 1:05 samples were close to the commercial membrane's, because there was no statistically significant difference between them (p > 0.05) [Table 1]. This value represents the characteristic of a membrane which is hardly wetted by water, as illustrated in [Figure 1].
|Table 1: Hydration Energy (ΔG) to membranes of 1:05, 1:10, and commercial membrane|
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The 15 layers of resin membranes 1:05 samples had a resistance value statistically superior to the commercial membrane's (p < 0.05).
The mechanical tests also revealed a difference in the test and commercial membrane in relation to rupture [Figure 5]. The burst effect observed in the tensile test for the test membrane is perpendicular to the direction of charging, while for the commercial membrane the deformation occurs in the same direction as that of the application of the force. The values obtained from the tensile tests are shown in [Table 2].
|Figure 5: Behavior of the membrane at the time of rupture. (a) 15 layer resin membrane and (b) Commercial sample|
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|Table 2: Values of tensile strength and elongation on maximum load to samples of 15 layer resin membrane 1:05 and commercial membrane|
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| Discussion|| |
The composition and structure of the membrane are important factors concerning the periodontal regeneration techniques. Moreover, properties such as outside topography, matrix stiffness, hydrophobicity, and surface chemistry play an important role in directing and the differentiation of stem cells. In this scope, chitosan has been used to generate a wide range of membranes with various morphological parameters and manufacture methods.
Chitosan shows good biocompatibility and an appropriate degradation and appears to have no toxicity activities, other than the bacteriostatic properties.,, The chitosan membrane was easy to manipulate and had a porous structure. These properties make it a promising material in the GTR procedure.,,, However, other studies showed that the chitosan membrane was rigid, degraded slowly, and was not well integrated with the surrounding soft tissue, indicating that this membrane was not suited to be used as a barrier membrane. This study developed a nylon-chitosan membrane with favorable physical properties and a low cost compared to a commercial nondegradable expanded poly. The results of this study showed that a nylon membrane covered by 1:05 of chitosan with 15 layers improved the ultimate tensile strength (UTS) by 464.04% and hydration energy by 19.22 mJ/m2.
The porous structure in the sublayers of the membrane appears to promote cellular adaption, sufficient permeation of nutrients, and a controlled growth factor for tissue regeneration. Microscopic analysis performed of the membranes' surfaces in this study showed a partial filling of the holes present among the nylon mesh wires, suggesting a selective permeability of the tested membrane [Figure 2] and [Figure 3]. This property is essential for bone regeneration. In this study, the 15 layer resin membrane 1:05 shows a high wettability due to the hydrophilicity and the porous aspect [Figure 1]b and [Figure 4]a.
The area where there is a greater wetting of the membrane by water is characterized by achieving higher Δ G values and the drop of solution appears more spread out on the surface being analyzed [Table 1]. It is desired that a partial wetting be present in GTR membranes for water since it is the conductive medium to carry nutrients to the area that needs to be regenerated. As high levels of water can compromise the mechanical memory of the membrane which can soften and losen their physical characteristic. However, the results in this study did not show any differences between the tested membrane (15 layers1:05) and the commercial membrane [Table 2].
The pure chitosan membrane adsorbed water very slowly owing to its high hydrophobicity. In one study, the chitosan membrane, which was fabricated with a porous microarchitecture, showed space for vascularization and, consequently, this property may be helpful in cell migration and bone regeneration.
The tensile test is a good indicator of material for use during the surgical procedure because it influences the flexibility, bending, preparation, and cut of membranes by the dentist. The strength and elongation on maximum load of the tested membrane tended to decrease with the presence of chitosan [Table 2]. The resistance of the membranes obtained with the deposits of chitosan (15 layers1:05) was statistically higher than the commercial membrane (p = 0.0079). This result can be considered positive, since it shows that there was a chemical interaction between the nylon fibers and chitosan, suggesting a good adhesion between the film and substrate. Another study also indicated the existence of an inter-molecular interaction between the chitosan and nylon. Indeed, subsequent trials should be conducted to assess the degree of adherence to the nylon resin matrix. The tensile tests were similar to the nylon mesh tensile strength values observed in literature and were favorable to the proposed 15 layer resin membrane 1:05, once its deformation was three times lower than the commercial membrane. This fact is a positive because it gives to the dentist a greater security in clinical usability. Besides, the rupture strength test for the tested membrane was perpendicular to the direction of loading while the commercial membrane deformation occurred in the same direction as that of the application of force.
Given the information discussed above, the membrane developed in this study shows some favorable properties that qualify it as a material that can be used in regenerative procedures using barriers, but biocompatibility studies, histologic observations, and histomorphometric analyses are necessary to indicate this membrane as a nonresorbable barrier in the GTR technique.
| Conclusion|| |
This in vitro experiment demonstrates that the tensile strength, micromorphological surface and wettability of the nylon/chitosan membrane fulfill the physics requirements to be used as a barrier in the GTR procedure. Furthermore, new in vitro and in vivo studies are necessary to use this nylon-chitosan as a framework for biomedical indication.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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Dr. Vanara F Passos
Monsenhor Furtado St., Fortaleza-CE 60430-170
Source of Support: None, Conflict of Interest: None
[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]
[Table 1], [Table 2]
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