10.59987/ads/2024.1.9-16
This study evaluates biocompatibility and biodegradation properties of PCL/Chitosan/BG-NP/Tetracycline and compares them with commercially available SureDerm membrane in Sprague Dawley Rats.Two different membranes (PCL/Chitosan/BG-NP/Tetracycline membrane and SureDerm membrane) were randomly inserted into subcutaneous pouches in the backs of 54 Sprague Dawley rats. The animals were sacrificed at day 7, 21, and 63. Biocompatibility properties (number and distribution of inflammatory cells, necrosis, neovascularisation, fibrosis, fatty infiltrate, tissue integration, tissue ingrowth) was assessed according to DIN EN ISO 10993-6. Biodegradation property was assessed macroscopically to check for membrane degradation rate. In terms of biocompatibility properties, the test membrane had overall irritancy score of 0 at day 7 and 21, and 0.66 at day 63, and it is considered non-irritant. Biodegradation of test membrane is faster compared to SureDerm at day 7 and 21. However, there is no significant difference between the two membranes at day 63. The GTR membrane PCL/Chitosan/BG-NP/Tetracycline is a good membrane with comparable biocompatibility and biodegradation properties to the SureDerm membrane.
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In Vivo Biocompatibility and Biodegradation Test of Two Barrier Membranes for Guided Tissue Regeneration
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Department of Biomedical and Anatomy, School of Medicine and Health Sciences, Atma Jaya Catholic University of Indonesia, Pluit Raya No. 2, North Jakarta, 14440
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Guided Tissue Regeneration
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In Vivo Biocompatibility and Biodegradation Test of Two Barrier Membranes for Guided Tissue Regeneration
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In Vivo Biocompatibility                                                                      Authors
                                                                                              Machiavelli Clavesia - School of Medicine

and Biodegradation Test of Two
                                                                                              and Health Sciences, Atma Jaya Catholic
                                                                                              University of Indonesia, Pluit Raya No. 2,
                                                                                              North Jakarta, 14440
Barrier Membranes for Guided                                                                  Stephani Dwiyanti - Department of
                                                                                              Dental Medicine, School of Medicine and
Tissue Regeneration                                                                           Health Sciences,
                                                                                              Atma Jaya Catholic University of
                                                                                              Indonesia, Pluit Raya No. 2, North
                                                                                              Jakarta, 14440
                                                                                              Veronika Maria Sidharta - Department of
Machiavelli Clavesia1                                                                         Histology, School of Medicine and Health
Stephani Dwiyanti2                                                                            Sciences,
Veronika Maria Sidharta3                                                                      Atma Jaya Catholic University
Dyonesia Ary Harjanti4                                                                        of Indonesia, Pluit Raya No. 2,
Mora Octavia2                                                                                 North Jakarta, 14440
Eko Adi Prasetyanto5                                                                          Dyonesia Ary Harjanti - Department
Tena Djuartina6                                                                               of Anatomical Pathology, School of
                                                                                              Medicine and Health Sciences, Atma
                                                                                              Jaya Catholic University of Indonesia,
1
  School of Medicine and Health Sciences, Atma Jaya Catholic                                  Pluit Raya No. 2, North Jakarta, 14440
University of Indonesia, Pluit Raya No. 2, North Jakarta, 14440
                                                                                              Mora Octavia - Department of Dental
2
  Department of Dental Medicine, School of Medicine and Health Sciences,                      Medicine, School of Medicine and Health
Atma Jaya Catholic University of Indonesia, Pluit Raya No. 2, North Jakarta, 14440            Sciences,
3
  Department of Histology, School of Medicine and Health Sciences,                            Atma Jaya Catholic University of
Atma Jaya Catholic University of Indonesia, Pluit Raya No. 2, North Jakarta, 14440            Indonesia, Pluit Raya No. 2, North
4
  Department of Anatomical Pathology, School of Medicine and Health Sciences,                 Jakarta, 14440
Atma Jaya Catholic University of Indonesia, Pluit Raya No. 2, North Jakarta, 14440            Eko Adi Prasetyanto - Department of
5
  Department of Pharmacy, School of Medicine and Health Sciences,                             Pharmacy, School of Medicine and
                                                                                              Health Sciences,
Atma Jaya Catholic University of Indonesia, Pluit Raya No. 2, North Jakarta, 14440
                                                                                              Atma Jaya Catholic University of
6
  Department of Biomedical and Anatomy, School of Medicine and Health Sciences,               Indonesia, Pluit Raya No. 2, North
Atma Jaya Catholic University of Indonesia, Pluit Raya No. 2, North Jakarta, 14440            Jakarta, 14440
                                                                                              Tena Djuartina - Department of
Corresponding author: Yohanes Eko Adi Prasetyanto                                             Biomedical and Anatomy, School of
E-mail: prasetyanto@atmajaya.ac.id                                                            Medicine and Health Sciences,
                                                                                              Atma Jaya Catholic University
Abstract                                                                                      of Indonesia, Pluit Raya No. 2,
                                                                                              North Jakarta, 14440
This study evaluates biocompatibility and biodegradation properties of PCL/Chi-
tosan/BG-NP/Tetracycline and compares them with commercially available Sure-
Derm membrane in Sprague Dawley Rats.Two different membranes (PCL/Chitosan/
BG-NP/Tetracycline membrane and SureDerm membrane) were randomly inserted
into subcutaneous pouches in the backs of 54 Sprague Dawley rats. The animals
                                                                                              License
were sacrificed at day 7, 21, and 63. Biocompatibility properties (number and distri-         This work is licensed under a Creative
bution of inflammatory cells, necrosis, neovascularisation, fibrosis, fatty infiltrate,       Commons Attribution-NonCommercial-
tissue integration, tissue ingrowth) was assessed according to DIN EN ISO 10993-              NoDerivatives 4.0 International License.
6. Biodegradation property was assessed macroscopically to check for membrane
degradation rate. In terms of biocompatibility properties, the test membrane had              Authors contributing to Annali di
                                                                                              Stomatologia agree to publish
overall irritancy score of 0 at day 7 and 21, and 0.66 at day 63, and it is considered
                                                                                              their articles under the Creative
non-irritant. Biodegradation of test membrane is faster compared to SureDerm at               Commons Attribution-NonCommercial-
day 7 and 21. However, there is no significant difference between the two mem-                NoDerivatives 4.0 International License,
branes at day 63. The GTR membrane PCL/Chitosan/BG-NP/Tetracycline is a good                  which allows third parties to copy and
membrane with comparable biocompatibility and biodegradation properties to the                redistribute the material providing
SureDerm membrane.                                                                            appropriate credit and a link to the
                                                                                              license but does not allow to use the
                                                                                              material for commercial purposes and to
Key words: Biocompatibility, Biodegradation, In Vivo, Polycaprolactone, Guided Tis-           use the material if it has been remixed,
sue Regeneration                                                                              transformed or built upon.

Introduction                                                                                  How to Cite
                                                                                              C Machiavelli, S Dwiyanti,VM Sidharta, D
Periodontitis is the main cause of tooth loss in adults. Research conducted in Jember Re-     Ary Harjanti, M Octavia,
gency for the period January-December 2014, several reasons for tooth extraction were         E Adi Prasetyanto, T Djuartina.
found, such as periodontitis (583 cases), caries (302 cases), impaction (58 cases), and       In Vivo Biocompatibility and
                                                                                              Biodegradation Test of Two Barrier
persistence (1 case) [1]. Periodontitis was responsible for the largest number of extracted   Membranes for Guided Tissue
tooth in all sociodemographic groups [1].                                                     Regeneration
Periodontitis affects many people throughout the world. According to the Ministry of          Annali Di Stomatologia, 15(1), 9-16.
Health of the Republic of Indonesia (Depkes RI), periodontitis ranks second in Indone-        https://doi.org/10.59987/ads/2024.1.9-16
sia [2].


10.59987/ads/2024.1.9-16                                                                                                   9
     In Vivo Biocompatibility and Biodegradation Test of Two Barrier Membranes for Guided Tissue Regeneration

     Periodontitis is a serious infectious disease and if not                Materials and Methods
     treated properly it can result in tooth loss [3]. The buildup
     of plaque bacteria on the surface of the teeth is the main              Research Design
     cause of periodontitis [3]. Plaque buildup initially causes
                                                                             This is an in vivo experimental research using Sprague
     gingivitis, which further develop into periodontitis result-            Dawley rats.
     ing in tissue damage periodontal support in the form of
                                                                             Ethical clearance for this research was granted prior to
     damage to the fibers, periodontal ligament and alveolar
                                                                             the commencement of the study by the Research Ethics
     bone. It can eventually cause mobility and tooth loss [3].
                                                                             Committee of the School of Medicine and Health Sci-
     One of the surgical treatment of periodontitis is carried
                                                                             ences Atma Jaya Catholic University of Indonesia. All
     out using the Guided Tissue Regeneration (GTR) [4].
                                                                             rats were kept under standard conditions in a purpose-
     This GTR method uses a barrier membrane which pre-
                                                                             designed room for experimental animals. They were
     vent epithelial tissue invasion and ensure the growth of
                                                                             treated according to the Animals in Research: Reporting
     periodontal ligament cells in the defect area [4]. GTR
                                                                             In Vivo Experiments guidelines for animal care, with free
     membrane should have a good biocompatibility, mechan-
                                                                             access to water and a standard diet.
     ical strength, biodegradability and antibacterial proper-
     ties. Barrier membranes are also useful for wound heal-
                                                                             Study Materials
     ing, isolation of gingival defects and clots stabilisation [5].
     Numerous investigations have focused on the develop-                    In this research, there were 2 types of membranes used:
     ment of resorbable materials for GTR membranes, ex-                     test membranes with the composition PCL/Chitosan/BG-
     emplified in the research conducted by Dikici et al [6].                NP/Tetracycline and commercially available SureDerm
     This research used polycaprolactone (PCL) which show                    as control membrane (Table 1).
     a good results because it has better biological properties              For negative control, the sham group was used. There
     than other polymers [6]. The PCL membrane also has                      were 9 groups in this study, namely;
     good mechanical, biocompatibility properties, and slow                  1. Positive Control 1: Rat inserted with SureDerm
     degradation rate [6]. This supports the use PCL as one                        membrane, terminated at day 7.
     of the composition for GTR membranes. However, our                      2. Positive Control 2: Rat inserted with SureDerm
     research combines two polymers so that the hydropho-                          membrane, terminated at day 21.
     bic nature of PCL can be overcome [6].                                  3. Positive Control 3: Rat inserted with SureDerm
     Chitosan is chosen as another polymer because it has                          membrane, terminated at day 63.
     good hydrophilic properties [7]. Chitosan also has an-                  4. Experimental Group 1: Rat inserted with PCL/Chi-
     tibacterial, anti-fungal, and wound healing properties                        tosan/BG-NP/Tetracycline membrane, terminated
     [8]. Another component, Bioactive Glass Nanoparticles                         at day 7.
     (BG-NP), is added to increase membrane stiffness. BG-                   5. Experimental Group 2: Rat inserted with PCL/Chi-
     NP also shows adequate extensibility in wet conditions                        tosan/BG-NP/Tetracycline membrane, terminated
     and is osteoconductive [9].                                                   at day 21.
     An alternative way to increase the antibacterial effect of a            6. Experimental Group 3: Rat inserted with PCL/Chi-
     membrane is with the help of antibiotics [10]. Several re-                    tosan/BG-NP/Tetracycline membrane, terminated
     searchers have succeeded in combining tetracycline into                       at day 63.
     a polymer solution to develop a membrane barrier [11].                  7. Negative Control 1: Sham group, no membrane in-
                                                                                   serted, but still undergo incision and suture, termi-
     Tetracycline is an effective bacteriostatic agent against
                                                                                   nated at day 7.
     many Gram-negative species including periodontopatho-
                                                                             8. Negative Control 2: Sham group, no membrane in-
     gens such as Aggregatibacter actinomycetemcomitans
                                                                                   serted, but still undergo incision and suture, termi-
     [12]. Tetracycline acts as a collagenase inhibitor, has
                                                                                   nated at day 21.
     anti-inflammatory action, bone resorption inhibitor, and
                                                                             9. Negative Control 3: Sham group, no membrane in-
     increases the attachment of fibroblasts to the root surface
                                                                                   serted, but still undergo incision and suture, termi-
     which increases periodontal tissue regeneration [12].
                                                                                   nated at day 63.
     Based on the description above, we fabricate barrier
     membranes containing PCL/Chitosan/BG-NP/Tetracy-
                                                                             Animals
     cline using electrospinning method. The objective of this
     research is to evaluate biocompatibility and biodegrada-                Fifty-four Sprague Dawley rats (mass, ± 100-300 g) were
     tion properties of PCL/Chitosan/BG-NP/Tetracycline and                  used in this study. Each group contained six rats allo-
     to compare them with commercially available SureDerm                    cated to each of 3 observation time points (7, 21, and
     membrane in Sprague Dawley Rats.                                        63 days).



     Table 1. Test and Control Article Descriptions
      Function                   Name                             Description                            Implant Sizes (mm x mm)
                                  PCL/Chitosan/BG-NP/                  PCL/Chitosan/BG-NP/
        Test Membrane                                                                                      10.0 x 10.0
                                  Tetracycline Membrane                Tetracycline Membrane
                                                                       Homologous acellular dermis
        Positive Control          SureDerm Membrane                                                        10.0 x 10.0
                                                                       and penicillin
        Negative Control          Sham Operation                       Without biomaterial insertion       10.0 x 10.0



10                                                                                                               10.59987/ads/2024.1.9-16
M. Clavesia et al.

Biocompatibility properties were tested according to the    Explantation and Biodegradation Test
DIN EN International Organization for Standardization
(ISO) 10993-6 standard for investigating the effects of     After trial periods of 7, 21, and 63 days, each group re-
subcutaneous implantation on local tissues. Assess-         ceived an overdose of Ketamine/Xylazine for euthana-
ment of biodegradation was done with the help of 10x10      sia. By adding a safety margin of approximately 5 mm
mm mica plastic, which was further divided into 9 small     on each side of the implanted membrane, a 20x20 mm
boxes. If the membrane covered <1/2 of the small box,       sample was removed from the back of the rat using a
a degradation value of 11.11% is given. If the membrane     scalpel, blade, and blunt scissors. Mica plastic was put
covered >1/2 of the small box, a degradation value of       on top of the biopsied sample to measure the degrada-
0% was given.                                               tion value of the membrane (Figure 2).


Subcutaneous Implantation                                   Fixation and Histopathological Staining
The rat was anesthetized intramuscularly using a sy-        The sample was placed in a 10% Neutral Buffered For-
ringe containing a combination of ketamine [90 mg/kg]       malin solution for 24 hours for fixation and were then
and xylazine [10 mg/kg]. The rat were then shaved and       sent for histological processing [13,15]. After fixation,
disinfected in the upper back area, and a transverse        each sample was dehydrated in a series of alcohol so-
incision was made on the rat’s backs with a scalpel. A      lutions of increasing concentration and subsequently
10x10 mm of GTR membrane was inserted into the back         embedded in paraffin [15]. The samples were cut into 4
subcutaneously [13] (Figure 1). The incision was closed     parts using a scalpel, after which they were further cut
with standard suture material (Prolene 6.0) [13]. Beta-     into 4 µm thick sections and stained with Hematoxylin
dine and gauze was applied on the wound. The rat was        and Eosin and Masson Trichrome for descriptive and
housed in separate cage until it was in stable condition,   semiquantitative histological evaluation [15].
after which the rats were moved back into their respec-
tive cages [13].                                            Semiquantitative Histological Analysis Ac-
                                                            cording 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× magni-
                                                            fication producing a score value indicating greater domi-
                                                            nance among them. The response of tissue-membrane
                                                            biological parameters is evaluated and assessed, as fol-
                                                            lows: (Table 2) [14].
                                                            1. The number and distribution of inflammatory cells
                                                                  semiquantitatively by looking at changes in neu-
                                                                  trophils, lymphocytes, plasma cells, macrophages,
                                                                  and giant cells as a function of distance from the
                                                                  material/tissue [14].
                                                            2. Presence and extent of necrosis [14].
Figure 1. Membrane Implantation into the Subcutaneous       3. Inflammatory response parameters (neovascu-
Back of Rats
                                                                  larization, capillaries with supporting fibroblas-
                                                                  tic 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 cap-
                                                            illary 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]. Ne-
                                                            crosis was evaluated, with the following grading system:
                                                            1, minimal; 2, mild; 3. moderate; 4, marked [15]. Fibro-
                                                            sis 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 us-
                                                            ing 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
     Figure 2. Macroscopic Aspects of Tissue Re-            cells, lymphocytes, plasma cells, macrophages, giant
     sponse to Different Membranes After Implantation       cells, necrosis which will then multiplied by two, then



10.59987/ads/2024.1.9-16                                                                                                     11
     In Vivo Biocompatibility and Biodegradation Test of Two Barrier Membranes for Guided Tissue Regeneration

     adding up with scores of neovascularization, fibrosis,                        Results
     and fatty infiltrate.
     The overall irritancy score of the test article at each                       Biocompatibility Test
     study time point was calculated as follows: Overall irri-                     Data of the biocompatibility test in the form of mean
     tancy score = test membrane irritancy score (PCL/Chito-                       histological evaluation for irritation/reactivity-cell type/
     san/BG-NP/Tetracycline membrane) – average irritancy                          respone according to ISO 10993-6 for day 7,21, and
     score of control membrane (Surederm membrane) If the                          63 is presented in Table 4,5, and 6. [15]. Lymphocyte
     result was a negative number, the irritancy score was                         cells, plasma cells, and macrophage cells were found
     considered to be 0.0. The irritancy grade was then deter-                     on the SureDerm membrane and PCL/Chitosan/BG-
     mined according to Table 3.                                                   NP/Tetracycline membrane on day 7 and day 21 (Table


     Table 2. Histological Evaluation System for Irritation/Reactivity – Cell Type/Response [13]
                                                                    Score (phf = Per High Powered (x400) Field)
                  Respone
                                             0                1                         2                         3                          4
                PMN cells                    0         Rare, 1-5/phf              6-10/phf                Heavy infiltrate               Packed
              Lymphocytes                    0         Rare, 1-5/phf              6-10/phf                Heavy infiltrate               Packed
               Plasma cells                  0         Rare, 1-5/phf              6-10/phf                Heavy infiltrate               Packed
              Macrophages                    0         Rare, 1-5/phf              6-10/phf                Heavy infiltrate               Packed
                Giant cells                  0         Rare, 1-2/phf               3-5/phf                Heavy infiltrate               Packed
                  Necrosis                   0            Minimal                   Mild                     Moderate                    Packed
            Neovascularization               0         Minimal capillary       Groups of 4-7               Broad band                Extensive band
                                                          proliferation        capillaries with            of capillaries             of capillaries
                                                        focal, 1-3 buds          supporting               with supporting            with supporting
                                                                                 fibroblastic                structures                fibroblastic
                Fibrocytes/                  0          Narrow band             Moderately                  Thick band               Extensive band
          fibroconnective tissue,                                               thick band
                  fibrosis
               Fatty infiltrate              0         Minimal amount          Several layers of            Elongated and              Extensive fat
                                                       of fat associated        fat and fibrosis         broad accumulation           surrounding the
                                                          with fibrosis                                  of fat cells about the           implant
                                                                                                              implant site



     Table 3. Irritancy/Reactivity Grade. Adapted from DIN EN ISO 10993-6 [13]
          Overall Irritancy Score                                                 Irritancy/Reactivity Status
          0.0 to 2.9                                                              Minimal or no reaction (non irritant)
          3.0 to 8.9                                                              Slight reaction (slight irritant)
          9.0 to 15.0                                                             Moderate reaction (moderate irritant)
          >15.1                                                                   Severe reaction (severe irritant)
     -     Irritation Score: (PMN Cells + Lymphocytes + Plasma Cells + Macrophages + Giant Cells + Necrosis) x 2 + (Neovascularization + Fibrosis
           + Fatty Infiltrate)
     -     Overall Irritation Score: PCL/Chitosan/BG-NP/Tetracycline Membrane Irritation Score – SureDerm Membrane Irritation Score



     Table 4. Mean Histological Evaluation for Irritation / Reactivity – Cell Type / Response According to ISO 10993-6 Guide-
     lines for Day 7
                                                                                             Neovas-                                   Tissue    Tissue
                          PMN     Lympho- Plasma            Macro-     Giant      Ne-                                    Fatty
                                                                                             cularisa-     Fibrosis                   Integra-     In-
                          Cell     cytes    Cell            phages     Cells     crosis                                Infiltrate
                                                                                               tion                                     tion     growth
         SureDerm                   0.5 ±        0.67 ±                                                      0.5 ±
                             0                                 0           0        0             0                          0           0         0
         Membrane                   0.84          1.21                                                       0.84
         Negative                                                                                           0.33 ±
                             0        0            0           0           0        0             0                          0           0         0
         Control                                                                                             0.52
         PCL/
         Chitosan/
                                    0.33 ±                  0.33 ±                                           1.17±
         BG-NP/              0                     0                       0        0             0                          0           0         0
                                     0.52                    0.52                                            1.33
         Tetracycline
         membrane



12                                                                                                                                10.59987/ads/2024.1.9-16
M. Clavesia et al.

4 & 5). On the other hand, there were no cells found                   irritant status of non irritant on day 7, day 21, and day
on day 7, day 21, and day 63 for the negative control                  63 (Table 7). Exemplary histological images stained with
(Table 4, 5, 6).                                                       Hematoxylin and Eosin as well as Masson Trichrome of
The PCL/Chitosan/BG-NP/Tetracycline membrane has a                     the three groups at day 7, 21, and 63 can be seen in
comparable irritation score as SureDerm membrane and                   Figure 3 and 4.


Table 5. Mean Histological Evaluation for Irritation / Reactivity – Cell Type / Response According to ISO 10993-6 Guide-
lines for Day 21
                                                                               Neovas-                                Tissue    Tissue
                  PMN      Lympho- Plasma         Macro-      Giant    Ne-                                Fatty
                                                                               cularisa-    Fibrosis                 Integra-     In-
                  Cell      cytes    Cell         phages      Cells   crosis                            Infiltrate
                                                                                 tion                                  tion     growth
 SureDerm                               0.5 ±      0.33 ±
                    0      1± 1.27                              0       0          0        3± 1.10         0           0           0
 Membrane                               0.84        0.82
 Negative                                                                                    2.67 ±
                    0          0           0          0         0       0          0                        0           0           0
 Control                                                                                      1.51
 PCL/
 Chitosan/
                             0.5 ±                  0.5 ±                                    2.33 ±
 BG-NP/             0                      0                    0       0          0                        0           0           0
                             0.55                   0.55                                      1.37
 Tetracycline
 membrane



Table 6. Mean Histological Evaluation for Irritation / Reactivity – Cell Type / Response According to ISO 10993-6 Guide-
lines for Day 63
                                                                               Neovas-                                Tissue    Tissue
                  PMN      Lympho- Plasma         Macro-      Giant    Ne-                                Fatty
                                                                               cularisa-    Fibrosis                 Integra-     In-
                  Cell      cytes    Cell         phages      Cells   crosis                            Infiltrate
                                                                                 tion                                  tion     growth
 SureDerm                                                                                    1.67 ±
                    0          0          0           0         0       0          0                        0           0          0
 Membrane                                                                                     1.86
 Negative                                                                                    1.67 ±
                    0          0          0           0         0       0          0                        0           0          0
 Control                                                                                      0.82
 PCL/
 Chitosan/
                                                                                             2.33 ±
 BG-NP/             0          0          0           0         0       0          0                        0           0          0
                                                                                              1.03
 Tetracycline
 membrane




Figure 3. Exemplary Histological Images Stained with He-               Figure 4. Exemplary Histological Images Stained with
matoxylin and Eosin of the Three Groups at Day 7, 21,                  Masson Trichrome for Fibrosis Evaluation of the Three
and 63.                                                                Groups at Day 7, 21, and 63
A. Negative Control, Day 7; B. Negative Control, Day 21; C. Nega-      A. Scoring 0, Negative Control, Day 7; B. Scoring 0, Negative Con-
tive Control, Day 63; D. SureDerm Membrane, Day 7; E. SureDerm         trol, Day 21; C. Scoring 1, Negative Control, Day 63; D. Scoring 2,
Membrane, Day 21; F. SureDerm Membrane, Day 63; G. PCL/Chi-            SureDerm Membrane, Day 7; E. Scoring 3, SureDerm Membrane,
tosan/BG-NP/Tetracycline Membrane, Day 7, H. PCL/Chitosan/BG-          Day 21; F. Scoring 4, SureDerm Membrane, Day 63; G. Scoring 3,
NP/Tetracycline Membrane, Day 21; I. PCL/Chitosan/BG-NP/Tetra-         PCL/Chitosan/BG-NP/Tetracycline Membrane, Day 7, H. Scoring 2,
cycline Membrane, Day 63                                               PCL/Chitosan/BG-NP/Tetracycline Membrane, Day 21; I. Scoring 3,
                                                                       PCL/Chitosan/BG-NP/Tetracycline Membrane, Day 63



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

     Biodegradation Test                                                       Discussion
     Based on the results of biodegradation tests, it can be                   Biocompatibility Test
     seen that the commercial SureDerm membrane was                            The GTR membrane has several criteria or requirements
     only slightly degraded on day 7 (27.5 ± 6.12%) and day                    to be called an ideal membrane. The first requirement is
     21 (28.33 ± 14.02%), while the PCL/Chitosan/BG-NP/                        to have biocompatibility properties [16]. Biocompatibility
     Tetracycline membrane has greater degradation on day                      is the ability of the material to adapt to the environment
     7 (50 ± 6.32%) and day 21 (50 ± 6.32%) (Table 7).                         where the membrane must not harm the body and have
     The percentage degradation test of SureDerm mem-                          non-toxic properties [16]. The membrane must not trig-
     brane and PCL/Chitosan/BG-NP/Tetracycline mem-                            ger the host’s immune system, sensitization, or chronic
     brane was compared using independent t-test for each                      inflammatory reactions [17]. Based on the results of the
     timeline. It was found that percentage degradation of the                 biocompatibility test, it can be seen that the GTR mem-
     two membranes were statistically significant for day 7                    brane with the composition (g) PCL/Chitosan/BG-NP/
     and day 21 in which PCL/Chitosan/BG-NP/Tetracycline                       Tetracycline = 11/0.5/0.5/0.04 has comparable irritancy
     membrane was degraded more quickly, but not signifi-                      score as the SureDerm commercial membrane and the
     cant for day 63.                                                          irritancy score overall non-irritant or non-irritating.
     Rate of degradation of the two membranes was faster                       This testing is in line with the research of Osathanon et al
     in the first 21 days, after which the degradation became                  [18]. Osathanon et al’s research discusses the biological
     more slowly (Figure 5).                                                   basis of GTR membranes in periodontal tissue healing

     Table 7. Mean Irritation Score After Membrane Implantation Day 7, Day 21, Day 63
                                                                                                          Overall
                                    Study Group                            Irritation Score                                 Irritation Status
                                                                                                     Irritation Score
                               SureDerm Membrane                              2.84 ± 0.87
                                  Negative Control                            0.33 ± 0.39
        Day 7                                                                                                0                 Non Irritant
                        PCL/Chitosan/BG-NP/Tetracycline
                                                                              2.49 ± 0.60
                                  membrane
                               SureDerm Membrane                              6.66 ± 0.87
                                  Negative Control                            2.67 ± 0.39
       Day 21                                                                                                0                 Non Irritant
                        PCL/Chitosan/BG-NP/Tetracycline
                                                                              4.33 ± 0.60
                                  membrane
                               SureDerm Membrane                              1.67 ± 0.87
                                  Negative Control                            1.67 ± 0.39
       Day 63                                                                                               0.66               Non Irritant
                        PCL/Chitosan/BG-NP/Tetracycline
                                                                              2.33 ± 0.60
                                  membrane

     Irritation Score: (PMN Cells + Lymphocytes + Plasma Cells + Macrophages + Giant Cells + Necrosis) x 2 + (Neovascularization + Fibrosis + Fatty
     Infiltrate)
     -      Overall Irritation Score: PCL/Chitosan/BG-NP/Tetracycline Membrane Irritation Score – SureDerm Membrane Irritation Score




                                                                                                                 Figure 5. Biodegradation Test
                                                                                                                 Chart



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M. Clavesia et al.

Table 8. Mean Biodegradation Test Data Post Membrane Implantation Day 7, Day 21, Day 63
                                                                               Biodegradation Percentage (%)
                    Sample
                                                        Day 0                  Day 7                     Day 21                Day 63

           SureDerm Membrane                               0               27.5 ± 6.12               28.33 ± 14.02          91.25 ± 7.50

   PCL/Chitosan/BG-NP/Tetracycline
                                                           0                 50 ± 6.32                 50 ± 6.32              95 ± 5.48
             membrane
                    P-value                                -                  <0.01**                    <0.05*                >0.05 ns
Note: ns: Non-Significant (p-value >0.05); * : Significant (p-value <0.05); ** : Very Significant (p-value <0.01)



and regeneration [18]. Bioactive GTR membranes have                            ideal criteria of a GTR membrane, but further observa-
been investigated and developed with the aim of creat-                         tions need to be made to determine the time needed for
ing membranes that not only act as a physical barrier                          all samples to be completely degraded.
but also induce biologics to enhance periodontal tissue                        Compared to SureDerm membrane, the test membrane
regeneration [18]. PCL has been introduced as a candi-                         is thinner. The SureDerm commercial membrane has a
date material for bioactive GTR membranes due to its                           thickness of 0.26 mm [23]. The test membrane has a
biocompatibility and simple fabrication procedures [18].                       thickness of 0.13 ± 0.03 mm [23]. A thicker membrane
Modification with other agents or biomolecules can be                          will have stronger mechanical strength than a thin one.
easily made [18]. PCL can be useful for promoting peri-                        Therefore SureDerm membrane degrade less rapidly
odontal tissue formation [18].                                                 due to its thicker thickness compared to the PCL/Chito-
Sarasam et al used a PCL/chitosan membrane which                               san/BG-NP/Tetracycline coated GTR membrane.
improves mechanical properties and cell viability com-
pared to pure chitosan [19]. Chitosan is a polysaccharide                      Research Limitations
that is much sought after in biomedical applications and
has been mixed with various macromolecules to reduce                           The research has limitations. This research model, us-
undesirable properties [19]. Dissolved chitosan and PCL                        ing a subcutaneous pouch of rats does not fully reflect
homogeneously in various mass ratios in a mixture of                           the conditions of the oral cavity and the oral microbi-
77% acetic acid in water and processed into uniform                            ome, so there could be variation in biocompatibility and
membranes [19]. Dynamic mechanical and thermal anal-                           biodegradation properties.
ysis shows that the crystallinity of PCL is suppressed                         The subcutaneous model is also less representative of
and its storage modulus is increased by the addition of                        the actual condition of GTR, in which the membrane
chitosan [19].                                                                 should be placed on a bone defect that has been filled
Another component of the membrane, bioactive glass is                          with bone graft, it is recommended that futher research
a promising material for tissue regeneration due to its                        can be carried out on more representative model, such
controlled degradability and ability to stimulate the for-                     as in bone defect on larger animals such as monkeys.
mation of new tissue [20]. Bioactive glass has demon-
strated excellent bioactivity and biocompatibility when                        Conclusion
implanted in bone defects [9]. Bioactive glass degrada-                        The GTR membrane PCL/Chitosan/BG-NP/Tetracycline
tion promotes osteogenesis by stimulating ions. stimu-                         composition (g) 11/0.5/0.5/0.04 is ideal as a GTR mem-
lates osteoconductivity [20]. PCL (polycaprolactone)                           brane because it has a good biocompatibility properties.
which is hydrophobic in nature was mixed with chitosan                         The PCL/Chitosan/BG-NP/Tetracycline membrane has a
which has hydrophilic properties [20]. PCL, known for its                      comparable irritation score as SureDerm membrane and
robust mechanical strength, can contribute to enhanc-                          irritant status of non irritant. The PCL/Chitosan/BG-NP/
ing the mechanical properties of chitosan, which tends                         Tetracycline membrane also has good biodegradation
to be inherently brittle [20]. A mixture of the two materi-                    properties which allows sufficient time for periodontal tis-
als shows good biocompatibility with each other because                        sue formation. However, the degradation rate is faster
PCL’s low melting point makes it easier to mix the two                         compared to SureDerm membrane.
polymers [19].
                                                                               References
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