Comparative evaluation of the physical properties of membranes for periodontal regeneration: An In vitro Study

Apoorva Mhatre; Devanand Shetty; Arvind Shetty; Suyog Dharmadhikari; Pooja Wadkar

doi:10.4103/aihb.aihb_113_21

ORIGINAL ARTICLE

Year : 2022 | Volume : 12 | Issue : 2 | Page : 151-158

Comparative evaluation of the physical properties of membranes for periodontal regeneration: An In vitro Study

Apoorva Mhatre, Devanand Shetty, Arvind Shetty, Suyog Dharmadhikari, Pooja Wadkar
Department of Periodontics and Oral Implantology, DY Patil University School of Dentistry, Navi Mumbai, Maharashtra, India

Date of Submission	31-Jul-2021
Date of Acceptance	29-Dec-2021
Date of Web Publication	13-May-2022

Correspondence Address:
Apoorva Mhatre
Department of Periodontics and Oral Implantology, DY Patil University School of Dentistry, Nerul, Navi Mumbai, Maharashtra
India

Source of Support: None, Conflict of Interest: None

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DOI: 10.4103/aihb.aihb_113_21

Abstract

Introduction: Barrier membranes are the devices used in guided tissue regeneration procedures to promote the repopulation of the wound space by periodontal ligament cells. Commercially available membranes have been used widely for periodontal regeneration. The aim of the present study is to compare and evaluate the physical properties of membranes used for periodontal regeneration. Materials and Methods: The tensile strength and surface topography using scanning electron microscope were analysed of three commercially available membranes: chorion membrane, Healiguide^™ membrane and PerioCol^®-GTR membrane were analysed. Results: The tensile strength of the PerioCol^®-GTR membrane was the highest, while that of the Chorion membrane was the least. Variations in the surface topography were observed in all three membranes groups. Conclusion: The study demonstrated variation in surface topography and tensile strength of the resorbable periodontal membranes, suggestive of differences in the time of resorption during regenerative periodontal procedures.

Keywords: Periodontal membranes, periodontitis, regeneration, surface topography, tensile strength

How to cite this article:
Mhatre A, Shetty D, Shetty A, Dharmadhikari S, Wadkar P. Comparative evaluation of the physical properties of membranes for periodontal regeneration: An In vitro Study. Adv Hum Biol 2022;12:151-8

How to cite this URL:
Mhatre A, Shetty D, Shetty A, Dharmadhikari S, Wadkar P. Comparative evaluation of the physical properties of membranes for periodontal regeneration: An In vitro Study. Adv Hum Biol [serial online] 2022 [cited 2023 Mar 30];12:151-8. Available from: https://www.aihbonline.com/text.asp?2022/12/2/151/345204

Introduction

Chronic periodontitis has been defined as “an infectious disease resulting in inflammation within supporting tissues of the teeth, progressive attachment loss and bone loss.”^[1] Periodontal flap surgery renders accessible root surface for both professional debridement and self-performed tooth cleaning after healing. Furthermore, periodontal surgery also aims to regenerate the periodontium that was lost because of periodontal disease.^[2]

Of all the possible outcomes of periodontal flap surgery, regeneration is the most favored one. Amongst all the approaches used for achieving regeneration, guided tissue regeneration is widely practised.^[3]

Guided tissue regeneration is based on the concepts given by Melcher in 1976.^[4] Melcher suggested that the cells that repopulate the root surface after periodontal surgery decide the nature of the attachment that would form. Only when the cells from the periodontal ligament proliferate the root surface first, the resultant outcome would be new connective tissue attachment and leading to periodontal regeneration.^[5]

Guided tissue regeneration has been defined by AAP Glossary of Periodontal Terms as “Regeneration of periodontal attachment through different tissue responses.” In the guided tissue regeneration approach, a barrier membrane is used to exclude the root surface from epithelial cells from repopulating, which leads to regeneration of the lost cementum, bone and periodontal ligament.^[6] Barrier membranes are the devices used to promote the repopulation of the wound space by periodontal ligament cells. In 1993, Gottlow classified membranes into three categories.^[7]

First-generation: Nonresorbable membranes
Second generation: Resorbable membranes
Third generation: Resorbable membranes with added growth factors.

Although the nonresorbable membranes provide good space maintenance and biocompatibility, the need for a second surgery after the initial surgery for the removal of membrane lead to discontinuation of these membranes and introduction of resorbable membranes.^[8] Collagen membranes are resorbable membranes derived from a natural source and are extensively used as barrier membranes.^[9]

Type I collagen-containing membranes have been fabricated and are made commercially available. The collagen membranes have the advantage of having good biocompatibility and biodegradability.^[10] These membranes also have some inherent bioactivity which plays an important role in natural remodeling.^[11] Thus, the aim of the present study is to compare and evaluate the physical properties of membranes used for periodontal regeneration.

Aim and objectives

To compare and evaluate the tensile strength of membranes commonly used for periodontal regeneration
To compare and evaluate the surface topography of membranes used for periodontal regeneration.

Materials and Methods

This comparative study was conducted on 21 specimens in our institute. The study duration was from July 2019 to October 2019. This study was done under the ethical guidelines of the Institutional Research and Ethical Committee. Since this study is in vitro, hence it did not require ethical clearance. Samples of resorbable periodontal regeneration membranes were included in the study, and non-resorbable membranes were excluded from the study.

Sample selection

In this study, three commercially available resorbable membranes, Chorion membrane, Healiguide membrane and PerioCol-GTR membrane, were included. They were divided into three groups consisting of seven samples each. Group 1 consisted of chorion membrane, Group 2 consisted of Healiguide and Group 3 consisted of PerioCol-GTR membrane.

The chorion membranes were acquired from Tissue Bank, Tata Memorial Hospital, Mumbai. The tissues were obtained during an elective cesarean section surgery of consenting mothers. The tissues underwent testing for infectious diseases and were terminally sterilised.

Healiguide^™ is a commercially available absorbable collagen membrane obtained from sheep. It comes as a thin sheet of high purity Type-I collagen membrane. It is purified by utilising American patented technology. Charge modification and slight calcification are carried out, which aids in guided tissue regeneration. The Healiguide^™ membrane is sterilised by ethylene oxide gas.

PerioCol^®-GTR is a commercially obtainable resorbable Type-I collagen membrane of fish origin. It is available as a pale white coloured membrane. The membrane is sterilised by gamma rays.

All seven specimens from each group were taken for sampling. Five membranes underwent tensile strength test, and two membranes underwent scanning electron microscope (SEM) analysis. The average dimension of the five Chorion membranes was 20 mm × 20 mm, that of five Healiguide^™ membranes were 20 mm × 30 mm and that of five PerioCol^®-GTR membranes were 25 mm × 30 mm.

Tensile testing procedure

The tensile strength test was carried out in the TPL department of Sasmira's Institute, Mumbai. Five specimens from each sample were taken. The test was carried out on a Universal testing machine of Tinius Olsen-UK, having a maximum capacity of 5000 N. The machine works on the principle of a constant rate of extension. The tensile strength test was carried out at a gauge length of 10 mm with a constant test speed of 50 mm/min. A longitudinal strain was put on the long length of the membrane specimen. The maximum load to break away the membrane was noted.

Scanning procedure

The SEM analysis was carried out at Icon Analytical Equipment Pvt. Ltd, Mumbai. The membranes were mounted on double-sided carbon tape. One blinded, trained and experienced examiner carried out the analysis of the membranes. The SEM used was FEI, Quanta 200. The membrane analysis consisted of the assessment of the surface topography of both the surfaces and the lateral surface of the membranes. Digital images were recorded by identifying the signals of the secondary electrons that were emitted from the specimen on exposure to an electron beam. The membrane specimens were analysed under a low vacuum with 65 Pa pressure. The membranes were analysed at × 100, ×500 and × 1000 magnifications for studying topography for the flat surface and at × 200, ×500 and × 1000 for the lateral surface.

Composition of membrane

The atomic composition of the membranes was conducted using a spectrometer and energy dispersive X-ray software EDX-METEX associated with the images of SEM (FEI Quanta 200). Different emissions were produced when there was an interaction between the electron beam and membrane samples. One of them was X-rays. The X-rays were absorbed by the detector of the dispersive energy, and the detector separated the characteristic X-rays of every element.

Statistical analysis

All the data that were collected were analysed and presented. The statical analysis was carried out using Stastical Product and Service Solutions (SPSS) version 21 for Windows (Armouk, NY:IBM corp) software. Statistical analysis was done using tools of descriptive statistics such as frequency and proportion (percentage) for representing categorical/nominal data. Quantitative variables were represented by mean and standard deviation. Probability P < 0.05 was considered as significant since the alpha error was set at 5%, with the confidence interval was set at 95% for this study. The power of the study was set at 80%, with a beta error set at 20%.

ANOVA F test was applied to compare intergroup measurements of tensile strength amongst three groups. Post-hoc data analysis, which follows the ANOVA F test, was done using Tukey's multiple comparison test was also used. Post-hoc test analyses multiple pairwise individual comparisons. The Chi-square test was used to find out the difference amongst the three groups in relation to surface topography.

Results

The mean tensile strength of the chorion membrane group was 0.977 ± 0.24 kgf, of the Healiguide^™ membrane group was 12.6 ± 2.7 kgf and that of PerioCol^®-GTR membrane group was 16.2 ± 3.25 kgf [Table 1]. [Graph 1] shows the descriptive statistics of tensile strength of the periodontal membranes in the three groups. The load needed to tear away the membranes was the least for the chorion membrane group and the highest for the PerioCol^®-GTR membrane group.

Table 1: Descriptive statistics of tensile strength (kg) of commonly used membranes (Group 1-Chorion membrane, Group 2-Healiguide^™, Group 3 – PerioCol^®) for periodontal regeneration

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One way ANOVA “F” test was applied across the data from the three groups of membranes to compare their tensile strength. [Table 2] displays the comparative statistics of tensile strength of all three groups. The one-way ANOVA “F” test indicated a highly significant difference within the three groups as well as amongst the three groups [Table 3]. [Graph 2] exhibits the pairwise comparative statics of tensile strength (kg) of all three membranes.

Table 2: Comparative statistics of tensile strength (kg) of all three membrane groups (Group 1-Chorion membrane, Group 2 Healiguide^™, Group 3 – PerioCol^®-guided tissue regeneration) using One way ANOVA F test

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Table 3: Pairwise comparative statistics of tensile strength (kg) of commonly used membranes (Group 1-chorion membrane, Group 2-Healiguide^™ membrane, Group 3-PerioCol^®-guided tissue regeneration membrane) for periodontal regeneration using Tukey's post-hoc test

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SEM analysis was performed, and the best images that were yielded by the microscope were selected. Both the surfaces of the chorion membrane group had an irregular surface. The membrane thickness was not uniform. Few circular pores were seen – the chorion membrane presented with an irregular lateral surface with several superimposed layers. The analysis of the lateral surface demonstrated oval and circular depressions with varying thickness present homogeneously in the membrane.

The Healiguide^™ membrane group displayed a smooth surface on both surfaces of the membranes. The membrane had a uniform thickness and was non-porous. Numerous structures exhibiting different forms and shapes were adsorbed on the surface of the membrane. The lateral surface of the membrane exhibited a smooth and uniform surface and displayed a single homogenous layer with very few vertical structures interspersed in between.

The PerioCol^®-GTR membrane group displayed smooth surfaces on either surface of the membrane. The membrane had a uniform thickness. Pores were not seen. Both the surfaces exhibited fibres of varying thickness organised in various directions. The interconnected fibres gave a mesh pattern appearance. There were very few structures adsorbed on the surface. The lateral surface showed an irregular surface with superimposition of many layers with very few vertical depressions that could be seen interspersed in between the layers.

[Figure 1] and [Figure 2] show the surface topography of the flat surface of the chorion membrane at ×100 and ×1000 magnification. [Figure 3] and [Figure 4] exhibit the lateral surface of the membranes of the chorion membrane group at ×200 and ×1000 magnifications. [Figure 5] and [Figure 6] show the flat surfaces of Healiguide^™ membrane at ×100 and ×1000 magnification. [Figure 7] and [Figure 8] show the lateral surface of the Healiguide^™ membrane at ×200 and ×1000 magnifications. [Figure 9] and [Figure 10] display the flat surface of the PerioCol^®-GTR membrane at ×100 and ×1000 magnification. The lateral surface of PerioCol^®-GTR membrane is seen at ×200 and ×1000 magnification in [Figure 11] and [Figure 12].

Figure 1: Chorion membrane ×100 magnification of flat surface. Pores can be seen on the flat surface.

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Figure 2: Chorion membrane ×1000 magnification of flat surface.

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Figure 3: Chorion membrane ×200 magnification of lateral surface. Irregular lateral surface with superimposition of different layers is observed.

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Figure 4: Chorion membrane ×1000 magnification of lateral surface. Oval and circular depressions of varying sizes can be seen.

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Figure 5: Healiguide^™ membrane ×100 magnification of the flat surface. Smooth and non-porous surface can be seen.

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Figure 6: Healiguide^™ membrane ×1000 magnification of flat surface. Numerous structures exhibiting different forms and shapes can be seen adsorbed on the surface of the membrane.

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Figure 7: Healiguide^™ membrane ×200 magnification of lateral surface. Smooth and non-porous lateral surface with a homogenous single layer can be seen.

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Figure 8: Healiguide^™ membrane ×1000 magnification of lateral surface. Very few vertical structures of different sizes interspersed in between can be observed.

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Figure 9: PerioCol^®-GTR membrane ×100 magnification of flat surface. Uniform smooth surface present with the interconnecting septae giving a mesh pattern appearance. Nonporous surface seen.

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Figure 10: PerioCol^®-GTR membrane ×1000 magnification of flat surface.

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Figure 11: PerioCol^®-GTR membrane ×200 magnification of lateral surface view. Superimposition of different layers seen.

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Figure 12: PerioCol^®-GTR membrane ×1000 magnification of lateral surface. Very few vertical depressions could be seen interspersed in between the layers.

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The data of each membrane for energy dispersive spectrometry weres demonstrated as percentages relative to weight. Variations were observed in the composition of the membranes. The composition of all three membrane groups is being mentioned in [Table 4]. Carbon, nitrogen and oxygen formed a major part of all the membranes. Calcium was found only in sheep-derived collagen membrane, Healiguide^™.

Table 4: Composition of all three groups of membranes (Group 1-chorion membrane, Group 2-Healiguide^™ membrane, Group 3-PerioCol^®-guided tissue regeneration membrane)

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Discussion

Guided tissue regeneration is affected by the physical properties of membranes, for instance, the stiffness, surface topography, porosity and chemical composition.^[12] Prevention or minimisation of inflammatory reactions, invasion of undesirable cells and maintenance of blood clots can be carried out by optimising membrane design and composition.^[13] Taking these things into consideration, the aim of this study was to compare and evaluate the physical properties of three commercially available membranes used for periodontal regeneration. The membranes were obtained from three different sources, human source, sheep derived and fish derived. The results from the analysis showed that there was a well-defined distinction amongst the three groups of membranes with respect to tensile strength as well as the surface topography. However, the base material Types I collagen for all the three membranes, differences could be observed in the tensile strength and surface topography. This difference can be mainly attributed to the different sources from which the membrane is derived. Furthermore, in the Healiguide^® membrane, slight calcification is carried out by the manufacturer to enhance guided tissue regeneration.

Significant differences were observed in the surface topography of the membranes when observed on SEM. In a study carried out by Raz, it was observed that the thickness of the membrane did not have a notable effect on the mechanical properties of the membrane.^[14]

Variations in the surface topography were observed in all three membranes groups. An in vitro study was conducted by Kasaj on three different collagen membranes where the ability of this membrane to endure the proliferation of osteoblastic cells, gingival fibroblasts and periodontal ligament cells was assessed.^[15] The study reported that, even though all the membranes were made up of collagen, there was a remarkable difference in the proliferation of cells. This study suggested that the variations in surface morphology and pore size of the membranes might account for the variance in their effect on the proliferation of the cells.

High tensile strength and stability of membranes are needed for combined horizontal and vertical augmentation of the ridge when comparatively larger defects have to be bridged, and there is a need to fix the membrane to the surrounding bone with pins.^[16],[17] Amongst the three groups of membranes, the fish-derived PerioCol^™-GTR membranes had the highest tensile strength, suggesting that their use can be expanded from usage for periodontal regeneration to horizontal or vertical ridge augmentation. In this context, the chorion membrane had the least tensile strength, and thus there were more chances of membrane collapse in the defect region when used for guided tissue regeneration as a barrier membrane. One reason for chorion membrane to have less tensile strength is that the resistance to longitudinal strain is offered only by the natural matrix.^[18] No external additives for strengthening the physical properties of the membrane are being added.

However, the chorion membrane finds its usage for root coverage procedures. Reports from a review by Chopra and Thomas stated that the foetal membranes had a degradation time of 4 weeks.^[19],[20] Thus, chorion membrane can be successfully used for root coverage procedures, where it can aid in regeneration. The usage of chorion membrane as a barrier membrane for intra bony defects should be done after careful case selection to prevent membrane collapse during wound healing.^[21]

When the membrane is used in vivo, as per the manufacturer's instruction, it has to be put in saline before its placement in the defect site. Studies have shown that more energy is required to tear away a dry specimen of the membrane than a wet specimen.^[22] Furthermore, it has been reported that considerable moistening can alter the mechanical properties of the membranes.^[14],[23] This implies that there would be a significant reduction in the tensile strength of the membrane when moistened, and thus careful handling of the membrane is required to prevent its inadvertent tearing. Therefore, it is essential to take note of the tensile strength of the membrane when used as a barrier for periodontal surgery.

In order to get long-term stable clinical results, it is necessary to study the physical properties of the periodontal regeneration membranes to avoid incidences of membrane collapse or membrane tear. The rationale for having superimposed layers in guided tissue regeneration membrane is established on the principle that different layers of the membrane can help in maintaining the structural, dimensional and mechanical properties for sufficient time to strengthen regeneration of periodontal apparatus. Furthermore, there is the presence of heterogeneous and non-uniform rates of degradation due to membrane superimposition.^[8] Therefore, additional research is needed for membrane fabrication and its structural superimposition of the currently available membranes.

Conclusion

The findings from this study would help the clinician in selecting the membranes for periodontal regeneration to achieve optimal clinical results. The physical properties of the membranes varied significantly from one another. The fish-derived PerioCol®-GTR membrane has the highest tensile strength when compared to the human-derived chorion membrane and sheep-derived Healiguide^™ membrane. The human-derived chorion membrane exhibited the least tensile strength, and hence further studies are required to be carried out to determine their effectiveness and usage in periodontal regeneration procedures. The study demonstrated variation in surface topography and physical properties of the resorbable periodontal membranes, suggestive of differences in the time of resorption during regenerative periodontal procedures. The findings from this study would also aid the manufacturers in improvising their product.

Limitations of the study

This study was carried out on sterile periodontal regeneration membranes. A study involving the effect of saliva on the physical properties would have been a better estimate for evaluating the tensile properties of the membranes.

Future possibilities

In the future, it would be helpful to analyse the effect that the oral environment can have on the physical properties of the periodontal regenerative membranes. In addition to this, a microbiological study of the periodontal regenerative membranes after incubation in saliva or serum to mimic intra-oral conditions would be of notable importance in this field.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

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