3- Non-shellfish chitosan from - - 社團法人中華牙醫學會 antibacterial GTR barriers J Dent Sci 2007‧Vol 2‧No 1 21 Preparation of non-shellfish sacchachitin and chitosan

J Dent Sci 2007‧Vol 2‧No1 19

Received: November 15, 2006 Accepted: February 27, 2007 Reprint requests to: Dr. Sheng-Yang Lee, Graduate Institute of Oral

Sciences, College of Oral Medicine, Taipei MedicalUniversity, No. 250, Wu-Hsing Street, Taipei, Taiwan11042, ROC.

Non-shellfish chitosan from the fruiting body residue of Ganoderma tsugae for long-lasting antibacterial

guided-tissue regeneration barriers

CHIEN-CHUNG CHEN1 LI-WEN CHEH1 JEN-CHANG YANG1 CHIH-MONG TSAI1, 2 EN-SHENG KEH1, 2 MING-THAU SHEU3 CHING-HUA SU4 HSIANG-HSI HONG5

CHE-TONG LIN1, 2 SHENG-YANG LEE1, 2

1 Graduate Institute of Oral Sciences, College of Oral Medicine, Taipei Medical University, Taipei, Taiwan, ROC.

2 Graduate Institute of Dentistry, College of Oral Medicine, Taipei Medical University, Taipei, Taiwan, ROC.

3 Graduate Institute of Pharmaceutical Sciences, College of Pharmacy, Taipei Medical University, Taipei, Taiwan, ROC.

4 Graduate Institute of Biomedical Materials, College of Oral Medicine, Taipei Medical University, Taipei, Taiwan, ROC.

5 Lin-Kou Medical Center, Chang-Gung Memorial Hospital, Lin-Kou, Taiwan, ROC.

Microbial control plays an important role in the clinical efficacy of dental practice. The purpose of this study was to develop a long-lasting antibacterial guided-tissue regeneration (GTR) barrier containing the non-shellfish chitosan from the fruiting body residue of Ganoderma tsugae. The powder of ground up G. tsugae fruiting body residue was processed to prepare the chitosan. The molecular weight, degree of deacetylation (DD), and yield of these non-shellfish chitosan derivatives were characterized. This material was also combined with other biomaterials to prepare polylactic acid (PLA)/Chitosan/ amorphous calcium phosphate (ACP) GTR barrier films for periodontal applications. By washing with deionized water, these GTR films were coagulated from the lactic acid solution. The film morphology was investigated using scanning electron microscopy, and the antibacterial function was also evaluated. The yields and DDs of the sacchachitin and chitosan were 39% ± 1% and 1% ± 0.2%, and 25% ± 2% and 68% ± 2%, respectively. Tests for chitosan against Actinobacillus actinomycetemcomitans showed that the antibacterial ability of the GTR barrier films, containing 2.08 wt% of the resultant chitosan, was 2.25 times higher, compared to 5 wt% doxycycline after the 8th day. The GTR barrier film maintained 56.58% of its antibacterial ability after the 18th day. We concluded that sacchachitin and chitosan from G. tsugae fruiting body residues possess excellent, long-lasting antibacterial function for potential dental applications. （J Dent Sci, 2(1)：19-29 , 2007）

Key words: Ganoderma tsugae, sacchachitin, chitosan, guided-tissue regeneration (GTR).

According to government statistics, approximately 80% of adults aged 35 years or older suffer from different degrees of periodontal diseases. Serious consequences can occur if proper and timely treatment is not performed. Guided-tissue regeneration (GTR)

is one of the most widely accepted treatment procedures for periodontal diseases. A thin barrier film is implanted between the soft and hard tissues to ensure proper tissue regeneration by separating and blocking the rapidly growing soft tissue from entering the bone defect and to ensure that there is space for the bone tissue to regenerate1-4. There are more than 15 commercial GTR products available (none produced locally), with only 8 of them being imported and available in the Taiwanese market. This was the main reason stimulating us to devote ourselves to this research.

C.C. Chen, L.W. Cheh, J.C. Yang, et al.


After implantation of a GTR film, there is at least a 3~6-week period of inflammation. To more-easily endure this stage, a daily antibacterial mouthwash or even antibiotic medicine has to be applied. Doxycycline in the Atrisorb-D® GTR barrier (Newtown, PA, USA) is one of the latest GTR barriers in the market, which is prepared by washing a lactic acid solution with water to precipitate a thin film. The active ingredient, doxycycline, a water-soluble chemical, can easily be washed away during the film formation process. Other experimental data showed that 90% of the doxycycline is released in the first 24 hours, leaving only 10% for the remaining period of inflammation. These data suggest that the current design using doxycycline does not have long-lasting antibacterial activity5.

To ensure effective and long-lasting antibacterial activity of GTR barrier films, we proposed taking advantage of a naturally occurring polysaccharide, chitosan, due to its proven antibacterial activity6-8,11,12. It is also well known that it has excellent wound- healing and osteoconduction activities10,21-26. It is also beneficial to use chitosan in such applications, since chitosan is mostly dissolved in acidic environments. Under normal saliva conditions, chitosan will not dissolve and wash away. Therefore, the antibacterial activity can be preserved for the desired GTR barrier.

Commercially, chitin and chitosan are produced from crustacean shells by chemical deacetylation and demineralization involving strong alkalis and acids, long periods of treatment, and high temperatures13. However, not only do the sources of the raw materials vary by area, species, and season, but the process is also costly and laborious. In addition, the physicochemical properties of chitin and its derivatives obtained by this method may be inconsistent14. In order to obtain chitin and chitosan of consistent quality, recent advances in fermentation technology have suggested that fungi can provide an alternate source of chitin and its derivatives, and they can be produced in a controlled environment year-round15,27. Furthermore, to prepare chitin and its derivatives, elimination of the deproteinization step and/or utilizing a shorter reaction time would certainly increase yields. Because plant proteins cannot serve as hosts for human pathogens, they are considered to be allergen-free and safer than their animal-based counterparts16-20. If these residual proteins can be removed during the harsh alkali treatment of the deacetylation step, considerable reduction in

production costs could be anticipated due to the reduction in NaOH usage and process times.

Ganoderma tsugae, called ling-zhi in Chinese, is an important fungus. The main components of G. tsugae fruiting bodies are β-1,3-D-glucan and chitin which were found to be safe materials in our previous studies 21-23. Our previous studies also showed that sacchachitin can enhance wound healing in Wistar rats and accelerate proliferation and migration of fibroblasts and keratinocytes with no cytotoxicity24. In human clinical trials, sacchachitin inhibited matrix metalloproteinases (MMPs) in impaired chronic ulcers, resulting in promotion of the healing process25. In the same model, a histoimmunological study de- monstrated that sacchachitin significantly increased type I collagen synthesis and fibroblast proliferation in the wound area26.

In this study, we tried to ensure the full utilization of G. tsugae and to produce both chitin and chitosan. We also tried to further improve the preparation pathway of sacchachitin and chitosan from the residue of G. tsugae fruiting bodies for higher yields with equally effective physicochemical properties. Through these optimized procedures, we hope to provide much-safer and stabler sources for the production of non-shellfish sacchachitin and chitosan as useful medical materials.

To demonstrate the antibiotic function of chitosan in an integrated system combined with such resorbable materials as polylactic acid (PLA) and amorphous calcium phosphate (ACP), we fabricated a GTR barrier film for use in periodontal surgery. The morphology, cell cytotoxicity, and antibiotic activities of these barrier films were characterized.

MATERIALS AND METHODS

Materials

From a factory in Nantou, Taiwan, the residue of the fruiting body of G. tsugae was collected after hot-water extraction. NaOH (1 N), 50% NaOH, 35% H2O2, lactic acid, and acetic acid were purchased from Acros Organics (NJ, USA). N/400 potassium polyvinylsulfate and 0.1% toluidine blue were obtained from Wako Pure Chemical (Osaka, Japan). Crustacean chitosan, with a degree of deacetylation of 90% and a molecular weight of 35 kDa, was obtained from Kiotek Corp. (Taiwan).

Long-lasting antibacterial GTR barriers

J Dent Sci 2007‧Vol 2‧No 1 21

Preparation of non-shellfish sacchachitin and chitosan

Ganoderma tsugae residue was deproteinized by treatment with 1 N NaOH at 100 °C for 5 hours. Bleaching of the resulting residue was carried out with 35% H2O2 at various pH values or separately with 0.1% hypochlorite. The product was finally washed with deionized water to remove the residual NaOH and H2O2. The pulp-like sacchachitin paste was further sieved and oven-dried to obtain solid films. Chitosan was prepared by alkaline deacetylation of G. tsugae residue and/or sacchachitin. It was then further purified by dissolving in a 2% lactic acid solution, followed by filtration to remove the insoluble materials. The soluble chitosan was precipitated with 1 N NaOH and washed with deionized water. The stepwise preparation pathway of sacchachitin and

chitosan is shown in Figure 1.

Preparation of GTR barrier films with chitosan

Several GTR barrier films with different com- positions were prepared by mixing PLA, chitosan, and ACP in a lactic acid solution. After washing the solution with deionized water, sample films were coagulated. Final drying was conducted in a vacuum oven for 24 hours. Control samples were also prepared with doxycycline instead of chitosan.

Morphological observation by scanning electron microscopy (SEM)

Dried sacchachitin and chitosan samples were loaded onto aluminum studs and coated with gold for

Figure 1. Preparation procedures of sacchachitin and chitosan from the residue of Ganoderma tsugae fruiting bodies.



3 minutes at 8 mA under a pressure of 0.1 torr. The morphology of each sample was scanned and examined using a Hitachi model S-2400 SEM.

Determination of the degree of deacetylation (DD) by infrared spectroscopy (IR)

IR spectra were recorded on a Perkin Elmer ATR spectrometer (Waltham, MA, USA) under dry air at room temperature. The DD (%) values of sacchachitin and chitosan were determined by the equation proposed by Baxter28:

]115*34501655[100% A

ADD −= ;

where A is the absorption of the IR intensity at a specific wave number.

Intrinsic viscosity and molecular weight

The chitosan was dissolved in 2% lactic acid at 40 °C for 4 hours. The intrinsic viscosity, η, was measured using an automated capillary viscometer (PVS1, Lauda, Germany). The operation was carried out at 25 °C. The K constant for the capillary was 0.009869 mm2/s2. Finally, the viscosity and average molecular weights, Mw, of the various chitosans produced were calculated using the Mark-Houwink equation29,30:

[η] = KMva

;

where K and a are the viscosity parameters.

Evaluation of antibacterial activity

The antibacterial activity of the chitosan was determined using Actinobacillus actinomycetemcomitans (ATCC 29523). The optical density (OD) of the sample was measured spectrophotometrically at 600 nm. One hundred microliters of the A. actinomycetemcomitans suspension was added to 800 µl of tryptic soy broth. The chitosan was dissolved in 2% lactic acid and then autoclaved at 121 °C for 15 minutes. Finally, an antibiotic test of A. actinomycetemcomitans was determined using 0.05%, 0.1%, 0.5%, 1%, and 2% chitosan solutions, and their OD values were measured after 5, 30, 60, 120 minutes, and 24 hours. Lactic acid (2%) was used as the control instead of the chitosan solution. The inhibitive percentage was calculated by the following equation:

%100OD

OD -OD (%) percentage Inhibitive

control

samplecontrol ×= .

The antibacterial activity of the GTR barrier films was evaluated similar to the above-described method with A. actinomycetemcomitans (ATCC 29523) after soaking in water for 0, 1, 3, 8, 11, and 18 days.

Statistical analysis

All data are shown as the mean ± standard deviation (SD) with n = 4 determinations. Means of the main effects were separated by the LSD multiple-range test using the SPSS software package (SPSS, Inc., Chicago, IL, USA).

RESULTS

Preparation of sacchachitin (SC) and chitosan and the bleaching process

The residue of G. tsugae fruiting bodies was first deproteinized with 1 N NaOH and then respectively subjected to sodium hypochlorite or H2O2 bleaching. After these processes, the dry weight of SC was measured. The yields obtained, as given in Table 1, were 26% ± 1.2% and 38% ± 1.6% after bleaching with sodium hypochlorite and H2O2, respectively. When the G. tsugae residue underwent bleaching with the 50% NaOH alkali preparation and then H2O2, its yield varied with the alkali preparation time. Two hours of processing time produced a yield of 38%, while 5 and 15 hours of processing time produced yields of 25% and 18%, respectively. The same samples were then dissolved in a 2% lactic acid solution and subjected to weak alkalosis and titration. The chitosan obtained from these processes was then subjected to oven-drying and weighing. However, not

Table 1. Yield of sacchachitin (SC) and chitosan produced from the residue of Ganoderma tsugae fruiting bodies

Final product Yield*

SC bleached with NaOCl 26% ± 1.2%

SC bleached with H2O2 38% ± 1.6%

Chitosan 1.1% ± 0.2%

* Mean ± S.D. (n=4).



all samples completely dissolved in the lactic acid solution.

Characterization of sacchachitin

The morphology of products prepared from the residue of G. tsugae fruiting bodies and sacchachitin, after being subjected to alkali deproteinization and H2O2 (or sodium hypochlorite) bleaching at various pH values, was observed by SEM. No significant difference (p > 0.05) in fiber diameters of the different samples was observed.

The physicochemical properties of chitin strongly depends on its DD. In the present study, the DD of sacchachitin is shown in Table 2. The DD was determined by IR spectroscopy in conjunction with CP-MAS 13C solid-state NMR31.

Characterization of chitosan

The DD and molecular weight of chitosan obtained from different alkali treatment times showed significant differences (p<0.05), as presented in Table 3.

Morphology, biocompatibility, and antibacterial test of the GTR barrier

The SEM micrographs revealed a micro-void morphology of the coagulated PLA film, as shown in Figure 2. A dense skin layer formed on top of the membrane, and the average pore size was less than about 5 μm along the cross-section. The results of the cytotoxicity tests of the GTR barrier films, listed in Table 4, showed reasonable cell viability.

Table 2. Degree of deacetylation (DD) for the residue of Ganoderma tsugae fruiting bodies and sacchachitin (SC)

Treatment DD (%)* Measured by IR

Residue of Ganoderma tsugae fruiting bodies 18.2 ± 2.3 a

Residue of the deproteinization process 18.7 ± 2.1 a

SC bleached with H2O2 at pH 7 23.2 ± 3.2 b

SC bleached with H2O2 at pH 9 24.4 ± 2.3 b

SC bleached with H2O2 at pH 11 25.3 ± 1.3 c

SC bleached with NaOCl 28.2 ± 1.8 c

* Mean ± S.D. (n = 4) of DD determinations. Means of different samples within the same column, followed by the same letter do not significantly differ (p < 0.05).

Table 3. Degree of deacetylation (DD) and the molecular weight (Mw) of the chitosans prepared with different alkali-treatments and times

Deacetylation treatment DD (%)* measured by IR Mw (kDa)

5 hours; 50% NaOH 68.3 ± 2.9 a 208

5 hours; 1 N - 5 hours; 50% NaOH (isolated from SC) 70.1 ± 4.2 a 208

15 hours; 50% NaOH 74.6 ± 3.7 b 59

* DD determinations: Mean ± S.D. (n = 4). Means of different samples within the same column followed by the same letter do not significantly differ (p < 0.05).



(a) 25 % (b) 35 %

(c) 45 % (d) 55 %

Figure 2. Cross-section of a coagulated polylactic acid barrier with various solid contents.

Table 4. Cytotoxicity test of guided tissue regeneration barriers

Sample Cell viability (%)

Positive control (100 ppm phenol) 28.8 ± 4.4

Negative control (medium) 100.0

PLA/chitosan* (99.02/0.08) 98.3 ± 4.9

PLA/chitosan^ (99.02/0.08) 59.7 ± 5.5

* Commercial chitosan. ^ Chitosan prepared in our lab.



The antibacterial activity of the chitosan was assessed using A. actinomycetemcomitans. The inhibitive percentage of chitosan at a final concentration of approximately 2% was 13% after 120 minutes. With 24 hours of exposure, the inhibitive percentage was 36% (Figure 3a). The minimal concentration of chitosan with an inhibitory effect on the growth of A. actinomycetemcomitans was determined by preparing a range of dilutions with an exposure time of 120 minutes. A chitosan concentration of 0.1% could slightly inhibit A. actinomycetemcomitans, while a dilution of 0.05% had no inhibitory effect at all. Figure 3b compares the antibacterial properties of the residue of G. tsugae fruiting bodies after treatment for 5 hours in 50% NaOH to those of chitosan extracted from sacchachitin and crustacean chitosan found in the marketplace. Within 1 hour, the 3 substances failed to display any significant differences, with the inhibitive percentages falling between 6% and 7%. After 120 minutes, the chitosan from the 2 different processes produced an antibacterial effect. However, there were no significant differences. In contrast, the antibacterial effect of the crustacean chitosan was much higher, and this particular trend became even more apparent after

24 hours.

DISCUSSION

Hydrogen peroxide (H2O2) possesses an intense odor and is a very effective oxidizer. Therefore, it is frequently used as a bleaching agent. In an alkaline environment, the bleaching reaction rate of hydrogen peroxide accelerates as the temperature rises. As the result, the formed perhydroxyl anions, as shown in the chemical equation below, react with colored impurities in fibers, oxidizing them into water-soluble compounds:

H2O2 ＋ OH-

→ HO-

2 ＋ H2O.

The sacchachitin yield obtained from the H2O2 bleach was 10%~15% higher, suggesting that using hydrogen peroxide for the bleaching treatment not only can prevent pollution during the production process and avoid residues of hazardous chlorine left on the final product, but can also increase the yield of sacchachitin. Since the components of G. tsugae residues are highly complex, residues of minerals and polysaccharides remain after treatments with 1 N

Figure 3(a). Growth inhibitions of Actinobacillus actinomycetemcomitans of chitosan solutions (with 0.05%, 0.1%, 0.5%, 1%, and 2% chitosan content). Results are presented as the mean of 3 experiments.

Time (min)

Inhi

bitiv

e pe

rcen

tage

(%)



NaOH and 0.6% sodium hypochlorite (or 35% H2O2 ) to eliminate proteins and melanin. These results indicated that the deacetylation degree of the 2-hour sample was not sufficiently high. The yields of chitosan obtained from the 5- and 15-hour samples ranged from 0.8% to 1.3%, which are comparable to previously reported data of yields of chitosan from mushroom of 1.2% ~ 3.3%10.

Results from the IR spectrum clearly showed the 3 characteristic absorption peaks of chitin, of amide III at 1310 cm-1, amide II at 1555 cm-1, and amide I at 1652 cm-1, demonstrating clearly that chitin is the primary component of the residue of G. tsugae fruiting bodies. After undergoing another series of treatments (including a deproteinization process and bleaching), the entity of chitin was preserved. The DD values, calculated from the absorption ratio of the hydroxyl

band at 3450 cm-1 to that of the amide band at 1655 cm-1, of the residue of G. tsugae fruiting bodies (18.20% ± 2.34%) and those after the deproteinization process (18.66% ± 2.11%) showed no significant difference (p > 0.05) , but significantly differed from those after bleaching (p<0.05). Using H2O2 at various pH values for bleaching, the measured DD values of sacchachitin ranged from 23% to 25%, indicating that no clear difference was caused by the pH value. DD values of sacchachitin bleached with sodium hypochlorite were 26%~28% and were shown to be slightly higher than those with the H2O2 bleaching process. For the present, we assumed that the increase in the DD value was the result of the bleaching mechanism of sodium hypochlorite. The obtained DD results were similar to those previously reported, around 12.5%~24.2%32. It should be noted that the

Figure 3(b). Growth inhibition of Actinobacillus actinomycetemcomitans of 2% sample A (chitosan via 5 hours; 50% NaOH preparation), sample B (chitosan via 5 hours, 1 N 5 hours, 50% NaOH preparation) and sample C (crustacean chitosan). Results are presented as the mean of 3 experiments.

Inhi

bitiv

e pe

rcen

tage

(%)

Time (min)



yield of sacchachitin was much higher than that of chitin extracted from mushroom (Agaricus bisporus) waste32.

We noted that a longer alkali treatment time led to a higher DD value and a lower molecular weight. After treatment with a 50% NaOH solution for 5 hours, the chitosan obtained from the residue of G. tsugae fruiting bodies had DD values of 66%~68% and a molecular weight of 208 kDa. On the other hand, 15-hour treatment with a 50% NaOH solution produced a DD value of 74% and molecular weight of 58.8 kD. In contrast, after a 5-hour 1 N deproteinization process, the sacchachitin extracted from the residue of G. tsugae fruiting bodies was subjected to further deacetylation for 5 hours in 50% NaOH. This produced a chitosan with a DD of 67%~70% and a molecular weight of 208 kDa. These data, however, showed no significant differences (p > 0.05) from those of chitosan which was directly extracted after a 5-hour treatment in 50% NaOH (Table 3). Therefore, we suggest eliminating this step and proceeding directly to the use of a strong alkali preparation for extraction of chitosan from the residue of G. tsugae fruiting bodies. Our results were similar to those of No et al. who described how elimination of the deproteinization step in chitosan preparation should be considered in light of the particular usage of the final chitosan product33. The ranges of our DDs and molecular weights were also similar to those of fungal chitosan previously reported by Shimahara et al. with DDs of 65%~90%34-36 and molecular weights of

30~190 kD36. The DD and molecular weight are important criteria for use in chitosan applications. Chitosan with a high DD has a high positive charge and is more suitable for use as an antimicrobial agent and in food applications. A 0.1% concentration of chitosan slightly inhibited A. actinomycetemcomitans, while a dilution of 0.05% had no inhibitory effect at all. From these data, it is evident that a chitosan concentration higher than 0.1% can produce a more-favorable antibacterial effect. These results demonstrate a very clear correlation between the effectiveness of the antibacterial activity of chitosan and its DD and molecular weight. Accordingly, we should try to further reduce the molecular weight of the chitosan in order to raise its antibacterial activity and promote its medical applications in the future.

The pore morphology of the GTR suggests good cell occlusion by blocking the ingrowth of epithelial cells, as shown in Figure 2.

The results of the cytotoxicity test of the GTR barrier films, listed in Table 4, show reasonable cell viability. Although films with chitosan were not quite as biocompatible as the ones containing chitosan from crustaceans, their biocompatibility was still in an acceptable range.

The incorporation of the antibiotic, doxycycline, in liquid GTR barriers is believed to prevent bacterial colonization of the barrier and reduces the incidence of undesirable infections during GTR procedures. In an attempt to minimize the use of antibiotics, positively charged polysaccharide chitosan was

Table 5. Result of the antibiotic effectiveness of guided tissue regeneration barriers

Time (day) PLA/doxycycline PLA/chitosan/ACP *

(99.82 / 0.08 / 0.10) PLA/chitosan/ACP (97.82 / 2.08 / 0.10)

0 89.4 ± 4.0a 78.0 ± 5.4abc 81.1 ± 8.8b

1 89.4 ± 2.9ab 18.8 ± 4.0ab 59.3 ± 17.1a

3 78.6 ± 4.5ab 11.2 ± 9.7ab 56.4 ± 6.7a

8 23.3 ± 7.7ab 8.1 ± 8.2cx 52.4 ± 13.2ac

11 20.4 ± 9.9ab 12.3 ± 10.6cx 55.2 ± 8.8ac

18 18.6 ± 8.9ab 15.7 ± 5.8 cx 56.6 ± 11.6 ac

Data were analyzed by one-way ANOVA. superscripts of a, b, c, x, and y indicate p < 0.05, considered to significantly differ. * PLA, polylactic acid; ACP, amorphous calcium phosphate.



chosen as a replacement for the water-soluble doxycycline. To mimic the time-dependence of the antibacterial properties, both the PLA/chitosan/ACP and PLA/doxycycline membranes were coagulated and soaked in deionized water for various time periods, and then the effectiveness of the antibacterial properties were measured. As listed in Table 5, up to day 3, the antibacterial activity of the PLA/ doxycycline (88.85/11.15) barriers was 78.6% ± 4.5%, but this had dropped to 23.3% ± 7.7% by day 8. On the other hand, the antibacterial activity of PLA/ chitosan/ACP (97.82/2.08/0.10) was 56.4% ± 6.7% on day 3, 52.4% ± 13.2% on day 8, and 56.6% ± 11.7% on day 18. Unlike doxycycline which is gradually released due to diffusion, the long-lasting antibacterial properties were implemented in this PLA/chitosan/ ACP GTR system. Even under regular antibiotic treatment and tooth cleaning for 4~6 weeks, Sanctis et al. showed bacterial colonization on ePTFE barriers. A GTR barrier with prolonged antibacterial activity is more desirable than the controlled release of the antibiotic, doxycycline.

In summary, we developed a better process to produce chitosan from G. tsugae fruiting body residues with higher yields and desired properties, enhancing the overall commercial value of G. tsugae. Among these properties, the most attractive one is its adequate and prolonged antimicrobial activity. We further prepared a GTR barrier film to validate the above- mentioned implications and confirmed them with promising data. These data suggest that chitosan/ sacchachitin can have wider potential dental and medical applications.

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