In Vivo Biocompatibility and Biodegradation Test of Two Barrier Membranes for Guided Tissue Regeneration

Introduction

Periodontitis is the main cause of tooth loss in adults. Research conducted in Jember Regency for the period January-December 2014, several reasons for tooth extraction were found, such as periodontitis (583 cases), caries (302 cases), impaction (58 cases), and persistence (1 case) [1]. Periodontitis was responsible for the largest number of extracted tooth in all sociodemographic groups [1].

Periodontitis affects many people throughout the world. According to the Ministry of Health of the Republic of Indonesia (Depkes RI), periodontitis ranks second in Indonesia [2].

Periodontitis is a serious infectious disease and if not treated properly it can result in tooth loss [3]. The buildup of plaque bacteria on the surface of the teeth is the main cause of periodontitis [3]. Plaque buildup initially causes gingivitis, which further develop into periodontitis resulting in tissue damage periodontal support in the form of damage to the fibers, periodontal ligament and alveolar bone. It can eventually cause mobility and tooth loss [3]. One of the surgical treatment of periodontitis is carried out using the Guided Tissue Regeneration (GTR) [4]. This GTR method uses a barrier membrane which prevent epithelial tissue invasion and ensure the growth of periodontal ligament cells in the defect area [4]. GTR membrane should have a good biocompatibility, mechanical strength, biodegradability and antibacterial properties. Barrier membranes are also useful for wound healing, isolation of gingival defects and clots stabilisation [5]. Numerous investigations have focused on the development of resorbable materials for GTR membranes, exemplified in the research conducted by Dikici et al [6]. This research used polycaprolactone (PCL) which show a good results because it has better biological properties than other polymers [6]. The PCL membrane also has good mechanical, biocompatibility properties, and slow degradation rate [6]. This supports the use PCL as one of the composition for GTR membranes. However, our research combines two polymers so that the hydrophobic nature of PCL can be overcome [6].

Chitosan is chosen as another polymer because it has good hydrophilic properties [7]. Chitosan also has antibacterial, anti-fungal, and wound healing properties [8]. Another component, Bioactive Glass Nanoparticles (BG-NP), is added to increase membrane stiffness. BG-NP also shows adequate extensibility in wet conditions and is osteoconductive [9].

An alternative way to increase the antibacterial effect of a membrane is with the help of antibiotics [10]. Several researchers have succeeded in combining tetracycline into a polymer solution to develop a membrane barrier [11].

Tetracycline is an effective bacteriostatic agent against many Gram-negative species including periodontopathogens such as Aggregatibacter actinomycetemcomitans [12]. Tetracycline acts as a collagenase inhibitor, has anti-inflammatory action, bone resorption inhibitor, and increases the attachment of fibroblasts to the root surface which increases periodontal tissue regeneration [12].

Based on the description above, we fabricate barrier membranes containing PCL/Chitosan/BG-NP/Tetracycline using electrospinning method. The objective of this research is to evaluate biocompatibility and biodegradation properties of PCL/Chitosan/BG-NP/Tetracycline and to compare them with commercially available SureDerm membrane in Sprague Dawley Rats.

Materials and Methods

Study Materials

In this research, there were 2 types of membranes used: test membranes with the composition PCL/Chitosan/BG-NP/Tetracycline and commercially available SureDerm as control membrane (Table 1).

For negative control, the sham group was used. There were 9 groups in this study, namely;

1. Positive Control 1: Rat inserted with SureDerm membrane, terminated at day 7.
2. Positive Control 2: Rat inserted with SureDerm membrane, terminated at day 21.
3. Positive Control 3: Rat inserted with SureDerm membrane, terminated at day 63.
4. Experimental Group 1: Rat inserted with PCL/Chitosan/BG-NP/Tetracycline membrane, terminated at day 7.
5. Experimental Group 2: Rat inserted with PCL/Chitosan/BG-NP/Tetracycline membrane, terminated at day 21.
6. Experimental Group 3: Rat inserted with PCL/Chitosan/BG-NP/Tetracycline membrane, terminated at day 63.
7. Negative Control 1: Sham group, no membrane inserted, but still undergo incision and suture, terminated at day 7.
8. Negative Control 2: Sham group, no membrane inserted, but still undergo incision and suture, terminated at day 21.
9. Negative Control 3: Sham group, no membrane inserted, but still undergo incision and suture, terminated at day 63.

Animals

Fifty-four Sprague Dawley rats (mass, ± 100–300 g) were used in this study. Each group contained six rats allocated to each of 3 observation time points (7, 21, and 63 days).

Biocompatibility properties were tested according to the DIN EN International Organization for Standardization (ISO) 10993-6 standard for investigating the effects of subcutaneous implantation on local tissues. Assessment of biodegradation was done with the help of 10x10 mm mica plastic, which was further divided into 9 small boxes. If the membrane covered <1/2 of the small box, a degradation value of 11.11% is given. If the membrane covered >1/2 of the small box, a degradation value of 0% was given.

Subcutaneous Implantation

The rat was anesthetized intramuscularly using a syringe containing a combination of ketamine [90 mg/kg] and xylazine [10 mg/kg]. The rat were then shaved and disinfected in the upper back area, and a transverse incision was made on the rat’s backs with a scalpel. A 10x10 mm of GTR membrane was inserted into the back subcutaneously [13] (Figure 1). The incision was closed with standard suture material (Prolene 6.0) [13]. Betadine and gauze was applied on the wound. The rat was housed in separate cage until it was in stable condition, after which the rats were moved back into their respective cages [13].

Explantation and Biodegradation Test

After trial periods of 7, 21, and 63 days, each group received an overdose of Ketamine/Xylazine for euthanasia. By adding a safety margin of approximately 5 mm on each side of the implanted membrane, a 20x20 mm sample was removed from the back of the rat using a scalpel, blade, and blunt scissors. Mica plastic was put on top of the biopsied sample to measure the degradation value of the membrane (Figure 2).

Fixation and Histopathological Staining

The sample was placed in a 10% Neutral Buffered Formalin solution for 24 hours for fixation and were then sent for histological processing [13,15]. After fixation, each sample was dehydrated in a series of alcohol solutions of increasing concentration and subsequently embedded in paraffin [15]. The samples were cut into 4 parts using a scalpel, after which they were further cut into 4 μm thick sections and stained with Hematoxylin and Eosin and Masson Trichrome for descriptive and semiquantitative histological evaluation [15].

Semiquantitative Histological Analysis According to DIN EN ISO 10993-6

Semiquantitative histological analysis was performed on each subcutaneous slide scanned according to the area of interest, without overlap, and captured at 400× magnification producing a score value indicating greater dominance among them. The response of tissue-membrane biological parameters is evaluated and assessed, as follows: (Table 2) [14].

1. The number and distribution of inflammatory cells semiquantitatively by looking at changes in neutrophils, lymphocytes, plasma cells, macrophages, and giant cells as a function of distance from the material/tissue [14].
2. Presence and extent of necrosis [14].
3. Inflammatory response parameters (neovascularization, capillaries with supporting fibroblastic structures, degree of fibrous capsule fibrosis stained with Masson Trichrome staining, and fat infiltration) [14].

Measurements were scored according to ISO 10993-6 guidelines: 0, none; 1, slight; 2, moderate; 3, marked; 4, complete/severe [15]. Neovascularization was scored according to capillaries present: 0, none; 1, minimal capillary proliferation; 2, groups of 4–7 capillaries ; 3, broad band of capillaries; 4, an extensive band of capillaries [15]. Inflammation is descriptively assessed based on the number of macrophages, polymorphonuclear cells, lymphocytes, plasma cells, and giant cells present [15]. Graded using the following scoring system: 1, Rare, 1–5/phf; 2, 6–10/phf; 3. heavy infiltrate; 4, packed [15]. Necrosis was evaluated, with the following grading system: 1, minimal; 2, mild; 3. moderate; 4, marked [15]. Fibrosis was evaluated, with the following grading system 1, narrow band; 2, moderately thick band; 3, thick band; 4, extensive band [15]. Finally, fatty infiltrate is assessed using the following scoring system 1, minimum amount of fat; 2, several layers of fat; 3, elongated and widespread accumulation of fat cells; 4, extensive fat completely [15]. Irritation score is obtained by adding up scores of PMN cells, lymphocytes, plasma cells, macrophages, giant cells, necrosis which will then multiplied by two, then adding up with scores of neovascularization, fibrosis, and fatty infiltrate.

The overall irritancy score of the test article at each study time point was calculated as follows: Overall irritancy score = test membrane irritancy score (PCL/Chitosan/BG-NP/Tetracycline membrane) – average irritancy score of control membrane (Surederm membrane) If the result was a negative number, the irritancy score was considered to be 0.0. The irritancy grade was then determined according to Table 3.

Results

Biocompatibility Test

Data of the biocompatibility test in the form of mean histological evaluation for irritation/reactivity-cell type/respone according to ISO 10993-6 for day 7,21, and 63 is presented in Table 4,5, and 6. [15]. Lymphocyte cells, plasma cells, and macrophage cells were found on the SureDerm membrane and PCL/Chitosan/BG-NP/Tetracycline membrane on day 7 and day 21 (Table 4 & 5). On the other hand, there were no cells found on day 7, day 21, and day 63 for the negative control (Table 4, 5, 6).

The PCL/Chitosan/BG-NP/Tetracycline membrane has a comparable irritation score as SureDerm membrane and irritant status of non irritant on day 7, day 21, and day 63 (Table 7). Exemplary histological images stained with Hematoxylin and Eosin as well as Masson Trichrome of the three groups at day 7, 21, and 63 can be seen in Figure 3 and 4.

	PMN Cell	Lymphocytes	Plasma Cell	Macrophages	Giant Cells	Necrosis	Neovascularisation	Fibrosis	Fatty Infiltrate	Tissue Integration	Tissue Ingrowth
SureDerm Membrane	0	1± 1.27	0.5 ± 0.84	0.33 ± 0.82	0	0	0	3± 1.10	0	0	0
Negative Control	0	0	0	0	0	0	0	2.67 ± 1.51	0	0	0
PCL/Chitosan/BG-NP/Tetracycline membrane	0	0.5 ± 0.55	0	0.5 ± 0.55	0	0	0	2.33 ± 1.37	0	0	0

	PMN Cell	Lymphocytes	Plasma Cell	Macrophages	Giant Cells	Necrosis	Neovascularisation	Fibrosis	Fatty Infiltrate	Tissue Integration	Tissue Ingrowth
SureDerm Membrane	0	0	0	0	0	0	0	1.67 ± 1.86	0	0	0
Negative Control	0	0	0	0	0	0	0	1.67 ± 0.82	0	0	0
PCL/Chitosan/BG-NP/Tetracycline membrane	0	0	0	0	0	0	0	2.33 ± 1.03	0	0	0

Biodegradation Test

Based on the results of biodegradation tests, it can be seen that the commercial SureDerm membrane was only slightly degraded on day 7 (27.5 ± 6.12%) and day 21 (28.33 ± 14.02%), while the PCL/Chitosan/BG-NP/Tetracycline membrane has greater degradation on day 7 (50 ± 6.32%) and day 21 (50 ± 6.32%) (Table 7).

The percentage degradation test of SureDerm membrane and PCL/Chitosan/BG-NP/Tetracycline membrane was compared using independent t-test for each timeline. It was found that percentage degradation of the two membranes were statistically significant for day 7 and day 21 in which PCL/Chitosan/BG-NP/Tetracycline membrane was degraded more quickly, but not significant for day 63.

Rate of degradation of the two membranes was faster in the first 21 days, after which the degradation became more slowly (Figure 5).

Discussion

Biodegradation Test

Based on the biodegradation test results listed in Table 7, it can be seen that all samples were not completely degraded on days 7 and 21 therefore they act as good barrier and allow sufficient rate of periodontal tissue formation of 4–6 weeks [22]. The addition of BG-NP increase the stiffness of the membrane so that it does not decompose or degrade easily. BG-NPs caused a significant increase in membrane thickness and surface area [21]. Samples on day 63 was completely degraded and invisible to the naked eye. This shows that the test membrane has a degradation capability that meets the ideal criteria of a GTR membrane, but further observations need to be made to determine the time needed for all samples to be completely degraded.

Compared to SureDerm membrane, the test membrane is thinner. The SureDerm commercial membrane has a thickness of 0.26 mm [23]. The test membrane has a thickness of 0.13 ± 0.03 mm [23]. A thicker membrane will have stronger mechanical strength than a thin one. Therefore SureDerm membrane degrade less rapidly due to its thicker thickness compared to the PCL/Chitosan/BG-NP/Tetracycline coated GTR membrane.

Research Limitations

The research has limitations. This research model, using a subcutaneous pouch of rats does not fully reflect the conditions of the oral cavity and the oral microbiome, so there could be variation in biocompatibility and biodegradation properties.

The subcutaneous model is also less representative of the actual condition of GTR, in which the membrane should be placed on a bone defect that has been filled with bone graft, it is recommended that futher research can be carried out on more representative model, such as in bone defect on larger animals such as monkeys.

Conclusion

The GTR membrane PCL/Chitosan/BG-NP/Tetracycline composition (g) 11/0.5/0.5/0.04 is ideal as a GTR membrane because it has a good biocompatibility properties. The PCL/Chitosan/BG-NP/Tetracycline membrane has a comparable irritation score as SureDerm membrane and irritant status of non irritant. The PCL/Chitosan/BG-NP/Tetracycline membrane also has good biodegradation properties which allows sufficient time for periodontal tissue formation. However, the degradation rate is faster compared to SureDerm membrane.