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Carbohydrate Polymers 151 (2016) 790–802

Contents lists available at ScienceDirect

Carbohydrate Polymers

j ourna l ho me pa g e: www.elsev ier .com/ locate /carbpol

functional chitosan membrane with graftedpigallocatechin-3-gallate and lovastatin enhances periodontal tissueegeneration in dogs

or-Shiunn Leea, Chien-Chen Leeb, Hung-Pin Linc,d, Wei-An Shiha, Wan-Ling Hsieha,hern-Hsiung Laie, Yasuo Takeuchi f, Yi-Wen Chenb,∗

Graduate Institute of Oral Biology, School of Dentistry, National Taiwan University and National Taiwan University Hospital, Taipei 10048, TaiwanGraduate Institute of Clinical Dentistry, School of Dentistry, National Taiwan University and National Taiwan University Hospital, Taipei 10048, TaiwanDepartment of Dentistry, MacKay Memorial Hospital, Taipei 10449, TaiwanDepartment of Dentistry, School of Dentistry, National Taiwan University, Taipei 10048, TaiwanCollege of Life Science, Kaohsiung Medical University, Kaohsiung 80708, TaiwanDepartment of Periodontology, Graduate School of Medical and Dental Sciences, Tokyo Medical and Dental University, Tokyo 113-8549, Japan

r t i c l e i n f o

rticle history:eceived 1 February 2016eceived in revised form 1 June 2016ccepted 5 June 2016vailable online 6 June 2016

eywords:

a b s t r a c t

Currently used guided tissue regeneration (GTR) membranes are mainly used as a barrier to preventepithelial cells growth into defects before new bone formation. The aim of this study was to develop atri-layer functional chitosan (CS) membrane with epigallocatechin-3-gallate (EGCG) grafted on the outerlayer for bactericidal activity, and lovastatin was included in the middle layer for controlled release.Successful EGCG grafting was demonstrated using Fourier transform infrared spectroscopy and EGCGgrafting significantly enhanced adhesion and proliferation of human gingival fibroblasts. The release

eriodontitisuided tissue regenerationhitosanollagen membranepigallocatechin-3-gallateovastatin

duration of lovastatin reached 21 days. CS-Lovastatin1 produced the highest alkaline phosphatase activityand EGCG14-CS exhibited the best bactericidal activity against periodontopathic bacteria. Finally, theEGCG14-CS-Lovastatin1 membrane showed a higher percentage of bone regeneration than BioMend® andcontrol groups in one-walled defects of beagle dogs. These results suggest that the EGCG14-CS-Lovastatin1membrane has the potential to be used as a novel GTR membrane.

© 2016 Elsevier Ltd. All rights reserved.

. Introduction

Periodontitis is a chronic inflammatory disease caused byicroorganisms and calculus accumulation on the root surface,

esulting in destruction of connective tissues and alveolar bone (deablo, Chapple, Buckley, & Dietrich, 2009; Pihlstrom, Michalowicz,

Johnson, 2005). It is highly prevalent, affecting up to 90% ofhe global population, and is considered to be the major cause

f tooth loss in adults (Petersen & Ogawa, 2005; Pihlstrom et al.,005). The objectives of periodontal therapy are first to arrest dis-ase progression and second to regenerate lost periodontal tissues

∗ Corresponding author at: Graduate Institute of Clinical Dentistry, School of Den-istry, National Taiwan University and National Taiwan University Hospital, No. 1,hang-Te St., Taipei 10048, Taiwan.

E-mail addresses: leebs@ntu.edu.tw (B.-S. Lee), zionist35@hotmail.comC.-C. Lee), hungpin@gmail.com (H.-P. Lin), v79319@hotmail.comW.-A. Shih), r00450017@ntu.edu.tw (W.-L. Hsieh), chern@kmu.edu.tw (C.-H. Lai),akeuchi.peri@tmd.ac.jp (Y. Takeuchi), yiwenchen@ntu.edu.tw (Y.-W. Chen).

ttp://dx.doi.org/10.1016/j.carbpol.2016.06.026144-8617/© 2016 Elsevier Ltd. All rights reserved.

(Aljateeli et al., 2014). Traditional periodontal surgeries are oftenperformed to gain access to diseased root surfaces and remove localfactors such as plaque, calculus, and infected cementum. Surgi-cal techniques include gingivectomy, open flap debridement, andosseous surgery, which may offer probing depth reduction andgain of clinical attachment (Becker et al., 1988; Caton, Nyman, &Zander, 1980; Kaldahl, Kalkwarf, Patil, Molvar, & Dyer, 1996). How-ever, new attachment achieved by these procedures is usually aresult of repair followed by long junctional epithelium with lessor no new connective tissue attachment and cementum formation(Bowers et al., 1989). In contrast, guided tissue regeneration (GTR),in which a barrier membrane is used to prevent epithelial cells andgingival tissue reaching the denuded root surface, has been shownto regenerate supporting tissues of the tooth, including new alve-olar bone, periodontal ligament, and cementum (Buser, Brägger,Lang, & Nyman, 1990; Garrett, 1996). The ideal properties of a

GTR membrane include the ability to exclude unwanted epithe-lial cell growth into the defect, protect the underlying blood clot,and degrade in adequate time to maintain a space for periodontal

dx.doi.org/10.1016/j.carbpol.2016.06.026http://www.sciencedirect.com/science/journal/01448617http://www.elsevier.com/locate/carbpolhttp://crossmark.crossref.org/dialog/?doi=10.1016/j.carbpol.2016.06.026&domain=pdfmailto:leebs@ntu.edu.twmailto:zionist35@hotmail.commailto:hungpin@gmail.commailto:v79319@hotmail.commailto:r00450017@ntu.edu.twmailto:chern@kmu.edu.twmailto:takeuchi.peri@tmd.ac.jpmailto:yiwenchen@ntu.edu.twdx.doi.org/10.1016/j.carbpol.2016.06.026

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igament cells, osteoblasts, and cementoblasts to repopulate on theoot surface (Bunyaratavej & Wang, 2001; Qasim, Delaine-Smith,ey, Rawlinson, & Rehman, 2015). The traditional GTR membraneas mainly used as a barrier to prevent epithelial cell downgrowth

nto defects before new bone formation. Recently, a functional drugelivery membrane has been developed to control the release ofrugs or growth factors, thus enhancing new bone regenerationLee et al., 2016; Monteiro et al., 2015; Yamano et al., 2014). Con-rolled and sustained release of antibiotics and/or growth factorsas become a promising approach to control disease and enhanceone regeneration.

Chitosan (CS) is a natural cationic polysaccharide with bio-ompatible, biodegradable, and antimicrobial properties. It cane processed into membranes, gels, nanoparticles, scaffolds, andponge forms for drug delivery systems (Xu, Lei, Meng, Wang, &ong, 2012). CS particles have been reported to inhibit the growthf Aggregatibacter actinomycetemcomitans and Porphyromonas gin-ivalis, and modulate inflammatory responses in human gingivalbroblasts (HGFs) (Arancibia et al., 2013). A CS/bioactive glassanoparticle composite membrane has been prepared by a solventasting method and can be used as a GTR membrane in periodontalegeneration (Mota et al., 2012). A bovine serum albumin-loadedS membrane was fabricated by a modified freeze-gelation methodnd exhibited a multi-stage delivery property, which can be usedn the treatment of periodontal disease (Ho, Hsieh, Hsiao, & Thien,010). A CS nanofiber mesh has been prepared as a wound dressingith sustained release of gentamicin for a bactericidal activ-

ty (Monteiro et al., 2015). PLGA-lovastatin-chitosan-tetracyclineanoparticles were successfully prepared using a double emulsionethod and showed an effective controlled-release pattern, good

iocompatibility, superior osteogenic potential, and anti-bacterialctivity (Lee et al., 2016). A clinical study has reported that a CS gelith or without 15% metronidazole is effective for probing depth

eduction in patients with chronic periodontitis (Akncbay, Senel, &y, 2007). CS has been widely used in the field of biomedicine and

s a good substrate candidate for a drug delivery membrane.Green tea is one of the most popular beverages worldwide.

atechin is a natural substance extracted from green tea, andpigallocatechin-3-gallate (EGCG) represents about 59% of totalatechins (Cabrera, Artacho, & Giménez, 2006). EGCG is consideredo have protective effects against diabetes, hypertension, can-er, and cardiovascular diseases, because it possesses a variety ofharmacological functions such as anti-oxidative, anti-angiogenic,nti-inflammatory, and antimicrobial effects (Cao & Cao, 1999;ordon & Wareham, 2010; Tipoe, Leung, Hung, & Fung, 2007).aily intake of green tea has been reported to reduce probingepth and clinical attachment loss, and alleviate bleeding on prob-

ng (Kushiyama, Shimazaki, Murakami, & Yamashita, 2009). Aross-sectional study reported that consumption of ≥1 cup/day ofreen tea decreased the odds ratio for tooth loss (Koyama et al.,010). EGCG inhibits lipopolysaccharide-induced osteoclastic boneesorption in vitro and attenuates inflammatory bone loss of theouse mandibular alveolar bone in vivo (Tominari et al., 2015).

ocal delivery of green tea catechin has been used as an effec-ive adjunctive therapy along with scaling and root planing for thereatment of chronic periodontitis (Gadagi, Chava, & Reddy, 2013;attarki, Pushpa, & Bhat, 2013; Hirasawa, Takada, Makimura, &take, 2002). These findings suggest that the anti-oxidative andnti-inflammatory effects of EGCG would be advantageous for localrug delivery.

Statins are lipid-lowering drugs that have become a mainstayn the treatment of high cholesterol to prevent cardiovascu-

ar and cerebrovascular events related to increased lipid levelsNissen et al., 2005). Statins may stimulate bone formation byncreasing expression of the bone morphogenetic protein (BMP)-

gene in bone cells and inducing angiogenesis to provide a new

mers 151 (2016) 790–802 791

direction in the field of periodontal therapy (Kataria, Kaur, Parvez, &Maurya, 2014). In a rat model, topical application of statins has pre-vented periodontal tissue breakdown and exerts beneficial effectson alveolar bone recovery after ligature-induced alveolar boneresorption (Seto et al., 2008). A single high-dose of simvastatin hasbeen demonstrated to stimulate murine cranial bone apposition,particularly when delivered under an occlusive membrane (Thylinet al., 2002). The application of statins is associated with decreasedtooth loss in chronic periodontitis patients (Cunha-Cruz, Saver,Maupome, & Hujoel, 2006). In randomized clinical trials, locallyadministered simvastatin significantly improved the clinical out-comes of scaling and root planning in patients with type 2 diabetesand chronic periodontitis (Pradeep & Thorat, 2010; Pradeep et al.,2012; Pradeep, Rao, Bajaj, & Kumari, 2013). These results indicatethat local delivery of statins is effective to alleviate periodontitis,thus enhancing bone regeneration.

A traditional GTR membrane only serves as a barrier. The aim ofthis study was to fabricate a functional CS membrane with graftedEGCG and controlled release of lovastatin. Cell adhesion, cyto-toxicity, alkaline phosphatase (ALPase) activity, and antibacterialactivity of the functional CS membrane were evaluated. In addi-tion, the bone regeneration potential of the fabricated membraneswas examined in one-walled defects in beagle dogs.

2. Materials and methods

2.1. Fabrication of the EGCG-CS-Lovastatin membrane

All chemicals were purchased from Sigma–Aldrich (St. Louis,MO, USA) and used without further purification. The EGCG-CS-Lovastatin membrane consisted of three CS layers. The surface ofthe top CS layer was grafted with EGCG, and lovastatin was includedin the middle CS layer. The CS layers were prepared by the solventevaporation method. CS (85% deacetylated, MW = 190–310 kDa)was dissolved in 1 M acetic acid to prepare a 2 wt% solutionthat was spread on a glass dish. The solution was dried in anoven at 60 ◦C for 1 h to evaporate the solvent. NaOH (1 M) wasadded as a non-solvent of CS to neutralize the dried CS mem-brane. After 15 min, the membrane was stripped off and washedwith distilled water three times. To prepare the middle CS layer,lovastatin was dissolved in chloroform at a concentration of0.1 mg/�l, and the mixture was added to 1 ml CS solution. Thetop CS layer was grafted with EGCG through succinylation ofEGCG and an ethyl(dimethylaminopropyl) carbodiimide (EDC)/N-hydroxysuccinimide (NHS) reaction (Fig. 1) (Bruneel & Schacht,1994; Yousefpour, Atyabi, Vasheghani-Farahani, Movahedi, &Dinarvand, 2011). EGCG (7, 14, and 21 mM), succinic anhydride,and 4-dimethylaminopyridine at a molar ratio of 1:10:15 weredissolved in dimethyl sulfoxide and stirred for 24 h at room tem-perature. Then, EDC and NHS were added to the solution at a molarratio of 1:5:2 (EGCG:EDC:NHS). Subsequently, 1.5 ml of the solu-tion was spin-coated onto the prepared CS membrane for 48 h atroom temperature to graft the EGCG. The membranes were washedwith deionized water. The three CS layers were adhered by apply-ing 1 M acetic acid on the interfaces of the three layers to solubilizesuperficially. They were then pressed together and solidified byimmersion in 3 ml of 50% EtOH for 5 min. Finally, the EGCG-CS-Lovastatin membranes were washed with deionized water andstored in a 60 ◦C oven.

CS tri-layer membranes grafted with three concentrationsof EGCG were prepared for 3-(4,5-dimethylthiazol-2-yl)-2,5-

diphenyl tetrazolium bromide (MTT) and antibacterial assays.In these assays, lovastatin was not added, and the membraneswere referred to as EGCG7-CS, EGCG14-CS, and EGCG21-CS. CS tri-layer membranes including lovastatin but without EGCG surface

792 B.-S. Lee et al. / Carbohydrate Polymers 151 (2016) 790–802

ures fo

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Fig. 1. Schematic illustration of the proced

odification were prepared for measurement of in vitro lovastatinelease, MTT assay, and ALPase assay. The membranes contained.1, 0.5, 1, and 2 mg lovastatin per membrane area (cm2), and wereeferred to as CS-Lovastatin0.1, CS-Lovastatin0.5, CS-Lovastatin1,nd CS-Lovastatin2, respectively.

.2. Tensile testing

The tensile test was performed using CS tri-layer membranesithout EGCG or lovastatin, EGCG14-CS-Lovastatin1 membranes,

nd two commercial collagen membranes, BioGide® (Geistlich,olhausen, Switzerland) and BioMend® (Sulzer Calcitek Inc., Carls-

ad, CA, USA). The samples were cut into squares (1 × 1 cm). Thehicknesses of the tested membranes were measured using a dig-tal caliper (Model CD-6BS; Mitutoyo, Tokyo, Japan). The samples

ere first immersed in PBS (pH = 7.4) for 30 min. They were thenxed using a cyanoacrylate adhesive (Zapit; DVA, Anaheim, CA,SA) and subjected to tensile forces in a microtensile testingachine (Microtensile Tester; Bisco, Inc., Schaumburg, IL, USA) at

crosshead speed of 1 mm/min.

.3. Characterization of EGCG-grafted CS membranes

The surfaces were examined using attenuated total reflectance-ourier transform infrared (FTIR) spectroscopy (FTIR-4200; Jasconternational Co., Ltd., Tokyo, Japan). FTIR spectra was recorded by

ressing the membranes against a ZnSe ATR crystal with slow scan-ing and a normal slit width. The wavelength detection ranged from000 to 650 cm−1, and the resolution was set at 2 cm−1 with 100cans.

r EGCG immobilization on CS membranes.

2.4. Assessment of lovastatin release in vitro

For quantification of lovastatin, a UV–vis spectrophotometer(Agilent 8453; Agilent Technologies, Santa Clara, CA, USA) was usedat a wavelength at 248 nm. Lovastatin was dissolved in acetonitrileand PBS at a volume ratio of

B.-S. Lee et al. / Carbohydrate Polymers 151 (2016) 790–802 793

7-CS,

2M3s

Fig. 2. FTIR spectra of (a) EGCG, (b) CS, (c) EGCG

4-well culture dish. After 4 h, 1, 3, and 5 days of incubation, 20 �lTT solution was added to each well, followed by incubation for

h. Subsequently, the medium was aspirated and 200 �l dimethylulfoxide was added to dissolve the formazan crystals. The optical

(d) EGCG14-CS, and (e) EGCG21-CS membrane.

density at 570 nm (OD570) of the formazan solution was measuredwith a microplate reader (ELx 800; BIOTEK, Winooski, VT, USA).

For the MTT assay with CS-lovastatin membranes, primarycultures of human bone marrow-derived osteoblasts (HOBs)

794 B.-S. Lee et al. / Carbohydrate Polymers 151 (2016) 790–802

CS-Lov

wiysE2awCTbaa

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Fig. 3. Lovastatin (mg) release per cm2 of CS-Lovastatin0.1,

ere used. Alveolar bone explants were harvested after obtain-ng informed consent from five healthy patients (aged 20–40ears) who underwent odontectomy of the third molar. Permis-ion to collect alveolar bone explants was obtained from thethics Committee of the National Taiwan University Hospital (No.01105080RC). The membranes were cut to a diameter of 5 mmnd placed at the bottom of transwell inserts (6.5 mm in diameterith a pore size of 3.0 mm; Costar Transwell Permeable Supports,orning, NY, USA). Empty inserts served as the negative control.he inserts were transferred into 24-well culture plates, which hadeen seeded with HOBs at 2 × 104 cells per well, and allowed todhere overnight at 37 ◦C. MTT assays were performed at 1, 3, 5, 7,nd 9 days of incubation.

.6. In vitro antibacterial activity assay

To simulate the human oral environment, sterile pooled salivaas obtained by collecting whole saliva from healthy volunteersithout stimulation (flow rate; 0.25 ml/min; pH 7.3) (Gong et al.,

012). The pooled saliva was centrifuged at 2000 rpm for 15 min toemove cellular debris and oral microorganisms. The supernatantas collected and mixed with 2.5 mmol/l dithiothreitol (Sigma-ldrich) to reduce salivary protein aggregation. The mixture wasentrifuged again and filter-sterilized through disposable, sterile.22-�m filters (Nalge Nunc International, Rochester, NY, USA) andtored at −20 ◦C until use.

A. actinomycetemcomitans (ATCC 43719), Prevotella nigrescensATCC 33563), and P. gingivalis (ATCC 33277) were cultured inrain-heart infusion (BHI) broth (Oxoid Ltd., Basingstoke, UK) with

�g/ml hemin (Sigma, Poole, UK). The hemin stock solution wasrepared by mixing 50 mg hemin with 1 ml of 1 M NaOH, adding9 ml distilled water, and then autoclaving the solution. Manipu-

ations outside of an anaerobic chamber [80% (v/v) N2, 10% (v/v)2, and 10% (v/v) CO2] were minimized to avoid the toxic effect ofxygen. Similarly, all reagents were stored under oxygen-free con-itions. Bacteria were cultured at 37 ◦C for 2 days under anaerobic

astatin0.5, CS-Lovastatin1, and CS-Lovastatin2 membranes.

conditions until reaching an optical density at 600 nm (OD600) of0.2 which corresponded to 107 CFU/ml.

Four types of membranes (CS, EGCG7-CS, EGCG14-CS, andEGCG21-CS) were prepared at a size of 5 mm × 10 mm and placed inpetri dishes after 75% alcohol immersion for 5 min. Ultraviolet lightwas used to kill bacteria and evaporate the alcohol. To create a sali-vary pellicle on the surfaces of the membranes, 5 �l pooled sterilesaliva was dropped onto each membrane and incubated for 1 h at37 ◦C. Then, 5 �l of bacterial suspension (optical density = 0.2) wasdropped onto each membrane, followed by incubation for 1 h at37 ◦C incubator under anaerobic conditions. The membranes weretransferred to sterile 48-well plates, and 1 ml BHI broth was addedto each well, followed by incubation at 37 ◦C for 2 days. Finally,all membranes were removed and the OD600 was measured spec-trophotometrically.

2.7. ALPase assay

The ALPase assay was performed using CS, CS-Lovastatin0.1, CS-Lovastatin0.5, CS-Lovastatin1, and CS-Lovastatin2 membranes. Thecell culture conditions were similar to those of the MTT assay usingCS-Lovastatin membranes. ALPase activity of the tested membraneswas measured at 3-, 5-, and 7-days of incubation by quantifyingthe conversion of p-nitro-phenylphosphate to p-nitrophenol. Thefluorescence intensity was measured at an excitation wavelengthof 356 nm and emission wavelength of 458 nm. ALPase activitywas normalized to the total protein content of the cells, whichwas determined spectrophotometrically using a Micro BCA proteinassay kit (Thermo Scientific, Grand Island, NY, USA).

2.8. Animal experiments

Three healthy male 1.5-year-old beagle dogs, weighing approx-imately 10 kg, were used in this study. The protocol and procedureswere approved by the Animal Research Center of the NationalTaiwan University (No. 20120500). All surgical procedures were

B.-S. Lee et al. / Carbohydrate Poly

Fig. 4. The performance of cell adhesion and proliferation on CS membranes con-taining various EGCG or lovastatin concentrations. (a) MTT assay results of HGFadhesion onto various amounts of grafted EGCG at 4 h after cell seeding. (b) MTTassay results of HGF proliferation on various amounts of grafted EGCG at 1, 3, and5 days after cell seeding. (c) MTT assay results of HOB proliferation on CS membranescontaining various amounts of lovastatin at 1, 3, 5, 7, and 9 days after cell seeding.Significant difference was labeled as * (p < 0.05).

mers 151 (2016) 790–802 795

performed under general and local anesthesia in sterile conditions.General anesthesia was achieved by subcutaneous injection of15 ml Zoletil® 50 (Virbac, Carros, France), 0.2 ml Rompun® (Bayer,Leverkusen, Germany), and 0.5 ml atropine. Local anesthesia wasperformed with 2% lidocaine hydrochloride containing epinephrineat a dilution of 1:10,000. All second premolars in the maxilla wereextracted to assure adequate space for further defect creation, and200,000 U penicillin G was administered intramuscularly. After 8weeks of healing, the mucoperiosteal flap was elevated, and one-walled intrabony defects (4 × 5 × 4 mm: buccolingual, mesiodistal,and depth, respectively) were created on the mesial and distal sidesof maxillary bilateral first premolars using round and fissure burswith a sterile saline coolant. The residual cementum was com-pletely removed by scaling and root planing using Gracey curettes.Three beagle dogs including twelve surgical sites were randomlydistributed into three groups. In the first experimental group, thedefects were filled with a gelatin-based sponge (Gelfoam®; Phar-macia & Upjohn Co, Kalamazoo, MI, USA) and covered with anEGCG14-CS-Lovastatin1 membrane. In the second group, defectswere filled with the Gelfoam® sponge and a resorbable collagenmembrane (Biomend®; Sulzer Calcitek, Carlsbad, CA, USA). For thecontrol group, the defects were filled with the Gelfoam® spongeonly. The mucoperiosteal flap was sutured using resorbable sutureswith primary closure. All wounds were kept clean by topical appli-cation of 2% chlorhexidine gluconate three times a week for 8weeks. The animals were sacrificed at 8 weeks after surgery.

2.9. Micro-computed tomography (CT) analysis

Before fixation, tissue and bone blocks containing defects weretrimmed to the proper size for the analysis chamber of a micro-CTdevice (in-vivo micro-CT SkyScan1176; Bruker, Kontich, Belgium).The blocks were mounted on a turntable that could be shiftedautomatically in the axial direction. Six hundred projections wererecorded over 180◦ of object rotation (0.3◦). The X-ray shadow pro-jections were digitized at 1024 × 1024 pixels with 256 gradations (8bit). The spatial resolution obtained was 35.52 �m/pixel. The trans-verse plane of images that were perpendicular to the tooth axis waschosen. A selected area of 120 × 150 pixels = 22.71 (mm2), similarto the defect size, was defined and calculated from the bottom ofthe defect (notch) toward the coronal area for 120 pixels (∼4 mm).By summarizing the net volume of the regenerated alveolar bonefrom the integrated volume of the selected areas of the periodontaldefect using Image J software, the newly formed bone volume (BV)and total defect volume (Sculean, Chiantella, Windisch, Arweiler,Brecx & Gera) were calculated. The percentage of bone fill wasdetermined as BV/TV (%). A single examiner, who was blinded tothe group assignments, performed micro-CT and the following his-tomorphometric analyses.

2.10. Histological analysis

Tissue blocks with defects were prepared, fixed in 10% bufferedformalin, and rinsed in PBS. After decalcification in Plank-Rychlosolution for 1 week, the blocks were trimmed, dehydrated, and

Table 1Tensile strengths of CS and commercial GTR membranes. Significant differences areindicated by different letters (n = 6, p < 0.05).

Thickness (mm) Strengths (MPa)

CS tri-layer membrane 0.17 3.65 ± 0.00aEGCG14-CS-Lovastatin1

membrane0.17 1.67 ± 0.14b

BioMend® 0.43 0.91 ± 0.00cBioGide® 0.34 0.87 ± 0.07c

796 B.-S. Lee et al. / Carbohydrate Polymers 151 (2016) 790–802

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ig. 5. ALPase activity of HOBs on CS membranes containing various amounts of lova

mbedded in paraffin. Serial sections of 5 �m in thickness were pre-ared in the mesiodistal plane at intervals of 100 �m, stained withematoxylin and eosin, and then examined under a light micro-cope.

.11. Statistical analysis

Data are expressed as means ± standard deviation. Mean val-es were statistically analyzed using one-way analysis of variance.ost-hoc comparisons were made using the Tukey-Kramer multipleomparison tests when the p-value was less than 0.05.

. Results

.1. Tensile strengths of membranes

The average tensile strengths of CS tri-layer membranes,GCG14-CS-Lovastatin1 membranes, BioMend®, and BioGide®

ere 3.65, 1.67, 0.91, and 0.87 MPa, respectively (Table 1). AfterGCG grafting and lovastatin inclusion, the tensile strengths ofGCG14-CS-Lova1 membranes were reduced significantly but weretill significantly higher than those of BioMend® and BioGide®.oreover, the thickness of the EGCG14-CS-Lova1 membrane was

.17 mm which was thinner than that of BioMend® and BioGide®.

.2. FTIR spectroscopy

Fig. 2 shows the FTIR spectra of EGCG, CS, EGCG7-CS, EGCG14-S, and EGCG21-CS. The characteristic peaks of EGCG includedn ester C O (1689 cm−1), aromatic C C (1610 cm−1), alcohol

O (1341 cm−1), aromatic O H (1215 cm−1), and C OH alcohols1142 cm−1) [42]. CS contained stretching vibrations of O H and/or

H (3327 cm−1), C H (2875 cm−1), amide C O (1648 cm−1),mide II N H (1576 cm−1), C O (1027 cm−1), deformation vibra-ions of C H (1375 cm−1), and antisymmetric stretching of C O C

1150 cm−1) (Caroni, De Lima, Pereira, & Fonseca, 2012; Tang et al.,013). After EGCG grafting, stretching of carbonyl C O (1731 cm−1)as found in succinylated EGCG (Marcazzan, Vianello, Scarpa, &igo, 1999). Moreover, aromatic O H (1215 cm−1) belonging to

at 3, 5, and 7 days after cell seeding. Significant difference was labeled as * (p < 0.05).

EGCG could be identified (Fig. 2c–e). Furthermore, amide II N Hshifted from 1576 cm−1 to 1550 cm−1.

3.3. Release profile of lovastatin

Fig. 3 shows the release profile of lovastatin (mg) per cm2

from membranes. Burse release was noted during the initial 6 hin CS-Lovastatin0.5, CS-Lovastatin1, and CS-Lovastatin2 groups.In contrast to the continued release of up to 70 days from CS-Lovastatin2, the release from CS-Lovastatin0.5 and CS-Lovastatin1was up to 21 days. In the group with least inclusion of lovastatin(CS-Lovastatin0.1), the release began from the second day and onlylasted up to 7 days.

3.4. MTT assay

MTT assay results after 4 h of incubation were used to evaluateHGF adhesion onto membranes with different amounts of graftedEGCG (Fig. 4a). The control group using petri dishes exhibited sig-nificantly higher values than EGCG7-CS and EGCG14-CS groups. Inaddition, both EGCG7-CS and EGCG14-CS groups exhibited signif-icantly higher values than EGCG21-CS and CS groups. MTT assayresults after 1, 3, and 5 days of incubation were used to evaluate HGFproliferation (Fig. 4b). Among the experimental groups, EGCG14-CS demonstrated the highest cell viability followed by EGCG7-CS.The cell viability of the EGCG21-CS group was higher than that ofthe CS group at 1 and 3 days but lower than at 5 days. MTT assayresults after 1, 3, 5, 7, and 9 days of incubation were used to evaluateHOB proliferation on CS membranes containing various amounts oflovastatin (Fig. 4c). No significant difference was found on the firstday. However, the cell viability was generally decreased with theincrease of the lovastatin concentration on days 5, 7, and 9.

3.5. ALPase assay

The ALPase assay revealed no significant difference among the

tested groups at 3 days (Fig. 5). However, the CS-Lovastatin1 groupdemonstrated the highest ALPase activity at 5 and 7 days. The val-ues of the CS-Lovastatin1 group were significantly higher than thoseof control and CS groups at 7 days and the CS group at 5 days.

te Polymers 151 (2016) 790–802 797

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Fig. 6. Antibacterial activities of CS, EGCG7-CS, EGCG14-CS, and EGCG21-CSmembranes. EGCG -CS exhibited superior antibacterial activity against A. acti-

B.-S. Lee et al. / Carbohydra

.6. Antibacterial activity

Fig. 6 shows the antibacterial activity of CS, EGCG7-CS, EGCG14-S, and EGCG21-CS membranes against A. actinomycetemcomitans,. nigrescens, and P. gingivalis. EGCG14-CS exhibited superiorntibacterial activity to CS, EGCG7-CS, and EGCG21-CS. The bacterialoncentrations were transformed from OD value to CFU/ml.

.7. Histology

Tissue blocks containing mesial and distal artificial defects of theaxillary first premolars were harvested at 60 days after surgery.

agittal sections along the mesiodistal direction were preparednd processed for histopathological examination by routine hema-oxylin and eosin staining. There was neither inflammatory cellnfiltration nor foreign body giant cell reactions noted in all groups.vidence of new cementum deposition on the root surface and newone formation as well as fibrous connective tissue approximate tohe surgical defect were discernible in both EGCG14-CS-Lovastatin1Fig. 7a and b) and BioMend® (Fig. 7d and e) groups, in contrast tohe presence of fibrous tissue and new bone formation only in theontrol group (Fig. 7g and h). The magnified inset of Fig. 7a showslump-looking osteoblasts rimming along the trabecular surfacef the bone adjacent to the defect in the EGCG14-CS-Lovastatin1roup (Fig. 7c). However, in the BioMend® group, there were lesssteoblasts lining the new bone trabeculae, which exhibited a rel-tively flat appearance (Fig. 7f). There was a lack of evident newementum formation in the control group (Fig. 7i).

.8. Micro-CT analysis

Fig. 8 shows the surgical procedures of the animal experimentsFig. 8a–f) and results of micro-CT analysis (Fig. 8g–i). New bone for-

ation was observed in the defects of EGCG14-CS-Lovastatin1 andiomend® groups (Fig. 8g), and the rectangular frame was chosen

or bone density analysis (Fig. 8h). The highest percentage of newone formation was observed in the EGCG14-CS-Lovastatin1 group62.03%), followed by the BioMend® group (46.07%), and then theontrol group (42.32%) (Fig. 8i).

. Discussion

The purpose of placing membranes for guided tissue/boneegeneration is to cover the bone and/or periodontal ligament, thusemporarily excluding the epithelium and gingival connective tis-ue during the postsurgical healing phase. It can prevent epithelialell migration into the wound and facilitate the repopulation of cellsrom the periodontal ligament and bone. Conventional collagen

embranes only serve as barriers without additional functions. Inhis study, EGCG was grafted on the surfaces of CS membranes, andovastatin release was controlled through the membranes, makinghe membranes multi-functional.

EGCG has been shown to have antioxidant, anti-inflammatory,nticarcinogenic, and antimicrobial functions (Reygaert, 2014).uch antimicrobial effects have been observed against variousram-positive and -negative bacteria such as Escherichia coli,almonella spp., Staphylococcus aureus, and Enterococcus spp.Jigisha, Nishant, Navin, & Pankaj, 2012; Steinmann, Buer,ietschmann, & Steinmann, 2013). For periodontal bacterium suchs A. actinomycetemcomitans (Hara et al., 2012), P. nigrescensOkamoto, Leung, Ansai, Sugimoto, & Maeda, 2003), and P. gingivalisAsahi et al., 2014), EGCG also exerts inhibitory effects. Lovas-

atin can increase BMP-2 mRNA expression in osteoblasts and boneepair in rats (Ho et al., 2011; Mundy et al., 1999). Therefore, theembranes fabricated in this study were suitable for guided tis-

ue/bone regeneration.

14

nomycetemcomitans (a), P. nigrescens (b), and P. gingivalis (c), compared with CS,EGCG7-CS, and EGCG21-CS. The bacterial concentrations were shown as CFU/ml.Significant difference was labeled as * (p < 0.05).

798 B.-S. Lee et al. / Carbohydrate Polymers 151 (2016) 790–802

Fig. 7. Histological micrographs of mesio-distal sections of sites treated with the EGCG14-CS-Lovastatin1, Biomend® (d–f), and control (g–i) membranes in beagle dogs. (a)In the EGCG14-CS-Lovastatin1 group, neither residual materials nor evident inflammatory cell infiltration were noted. (b) New cementum (NC), new bone formation (NB),and fibrous connective tissue (FC) were observed in the notch area. (c) Magnified inset of (a) showed plump-looking osteoblasts rimming along the trabecular surface of thebone adjacent to the defect. (d) In the Biomend® group, no residual materials or evident inflammatory cell infiltration were noted. (e) New cementum, new bone formation( nifieds rmati new

waTbf

NB), and fibrous connective tissue (FC) were observed in the notch area. (f) The magurface of the bone adjacent to the defect. (g and h) In the control group, new bone fonset of (g) revealed new bone formation and fibrous connective tissue but a lack of

The tensile strength of the EGCG14-CS-Lovastatin1 membraneas significantly higher than those of BioMend® and BioGide®,

lbeit the EGCG -CS-Lovastatin membrane was thinner (Table 1).

14 1o increase the strength of the EGCG14-CS-Lovastatin1 mem-rane, we employed a solvent evaporation method instead of thereeze-gelation method. Therefore, dense structures rather than

inset of (d) shows osteoblasts with a flat appearance rimming along the trabecularion and fibrous connective tissue were observed in the notch area. (i) The magnifiedcementum formation.

porous structures were produced (Ho et al., 2004), and thesedense structures prevented the migration of epithelial cells. More-over, histological examination after the animal experiments did

not reveal remnants of membranes or inflammation, suggestingdegradation of the membranes (Fig. 7). The decrease in the ten-sile strength of the EGCG14-CS-Lovastatin1 membrane compared

B.-S. Lee et al. / Carbohydrate Polymers 151 (2016) 790–802 799

Fig. 8. Surgical procedures of the animal experiments (a–f) and the results of micro-CT analyses (g–i). (a) Soft tissue healing was good after extraction of maxillary secondpremolars. (b) Elevation of the mucoperiosteal flap. (c) One-walled defects were created at mesial and distal sides of maxillary first premolars. (d) Gelfoam® was placed in thedefects to make space. (e) EGCG14-CS-Lovastatin1 (left) and Biomend® (right) membranes were trimmed to the appropriate size and placed over the defects. (f) The woundwas closed by sutures with primary closure. (g) New bone formation was observed in defects of EGCG14-CS-Lovastatin1 (left) and Biomend® (right) groups. (h) The rectangularframe was chosen for bone density analysis. (i) The highest percentage of new bone formation was observed in the EGCG14-CS-Lovastatin1 group (62.03%), followed by theBioMend® group (46.07%) and the control group (42.32%). Significant difference was labeled as * (p < 0.05).

8 te Poly

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00 B.-S. Lee et al. / Carbohydra

ith that of CS tri-layer membranes was possibly attributed to thenclusion of lovastatin that decreased the structural integrity.

Succinic anhydride was used as a spacer to graft EGCG onto CSembranes. It first reacted with the hydroxyl groups of EGCG to

orm a carboxylic group, and the resulting succinyl EGCG formedn amide bond with CS through EDC and NHS reactions. After EGCGrafting, the appearance of three peaks including the aromatic O H1215 cm−1) of EGCG, carbonyl C O (1731 cm−1) of the succinylroup, and amide II N H (1550 cm−1) indicated successful graftingf EGCG onto CS membranes. In addition, the peak intensity wasncreased with the increase of EGCG concentrations (Fig. 2).

The release of lovastatin was controlled through inclusion inhe middle layer of the membranes. The purpose of the designas to reduce the burst release and prolong the release duration.lthough burst release still occurred during the initial 6 h (Fig. 3),S-Lovastatin0.5 and CS-Lovastatin1 groups released lovastatin forp to 21 days which is close to the duration (4–6 weeks) requiredo heal periodontal tissues. The 70 days of lovastatin release inhe CS-Lovastatin2 group might be more suitable for guided boneegeneration that requires longer stimulation by lovastatin.

ALPase activity assay revealed that the concentration of lovas-atin in the CS-Lovastatin1 membrane was optimal to stimulatesteogenic differentiation, and the lovastatin concentration in CS-ovastatin2 was too high (Fig. 5). Moreover, the ALPase activity wasncreased with the increase in culture time. In contrast, cell viability

as decreased at 5 days after seeding (Fig. 4c). A previous study hashown a reciprocal relationship between proliferation and differ-ntiation (Lian & Stein, 1992). Lovastatin might initially inhibit cellroliferation and then stimulate osteogenic differentiation-relatedene expression.

Cell adhesion has been evaluated using MTT assay at 4 h afterell seeding (Park, Lee, Lee, & Suh, 2003). Although CS exhibits

positive surface charge of protonated amino groups that act asn excellent mucoadhesive (Dash, Chiellini, Ottenbrite, & Chiellini,011), the CS membranes did not provide better HGF adhesionhan the other groups (Fig. 4a). It has been reported that theeta potential of a CS membrane equals zero under physiolog-cal conditions (pH 7.4) and thus loses the ability to adhere tolycosaminoglycans, fibronectin, and vitronectin (Lopez-Perez, dailva, Serra, Pashkuleva, & Reis, 2010). Modification of CS with inter-ediate polar and Lewis basic components enhances cell adhesion

nd proliferation (Lopez-Perez, Marques, da Silva, Pashkuleva, Reis, 2007). The polarities of the four major catechins arepigallocatechin (very polar) > epicatechin (polar) > EGCG (inter-ediate polar) > epicatechin gallate (non-polar) (Lai, Lee, Chang, &

iaw, 2008). The intermediate polarity of EGCG on CS membranesnhanced HGF adhesion. However, a high concentration of EGCGe.g. EGCG21-CS) might be too polar for HGF adhesion. A similar phe-omenon was also reflected by the cell viability of the EGCG14-CSroup at 3 and 5 days, which was better than that of the EGCG21-CSroup (Fig. 4b).

Two types of collagen membranes (Bio-Gide® and Biomend®)ere chosen to compare tensile strengths because of their dif-

erences in collagen types and cross-linking. Biomend® containsype I collagen from the bovine Achilles tendon, which resorbs in–8 weeks, and Bio-Gide® contains type I and III collagen fromorcine dermis with a resorption rate of 24 weeks (Bunyaratavej

Wang, 2001). These two membranes are commercially availablend widely used as guided tissue/bone regeneration membranes,nd have been demonstrated to reduce probing depth and gainlinical attachment level in cases with peri-implantitis and peri-dontitis when combined with autogenous bone or other bone

rafts (Bunyaratavej & Wang, 2001). The fact that the tensiletrength of the EGCG14-CS-Lovastatin1 membrane was higherhan that of Bio-Gide® and Biomend® membranes indicateshat the EGCG14-CS-Lovastatin1 membrane has better mechanical

mers 151 (2016) 790–802

properties that may provide effective space and an appropriatetime for new bone formation. To evaluate the bone regenerationpotential in dogs, we used the Biomend® membrane instead ofthe Bio-Gide® membrane for comparison because Biomend® canbe appropriately resorbed within 2 months. The one-walled intra-bony defect model is surgically created at mesial and distal sidesof maxillary first premolars. It is the preferred model to observethe nature of wound healing and evaluate the efficacy of biomate-rials following periodontal regeneration therapy (Kim et al., 2011).Moreover, we used Gelfoam® as a space-making material insteadof bone grafts because bone grafts may contribute to the peri-odontal regeneration and influence the comparison of the testedmembranes.

Histological analyses showed that all groups had new boneformation. The lack of inflammatory cells and giant cells reac-tions to foreign bodies demonstrated the biocompatibility ofthe tested materials. In contrast to the relatively decreasednumbers of rimming osteoblasts with a flat appearance in theBiomend® group, the plump-looking osteoblasts in the EGCG14-CS-Lovastatin1 group suggested better osteogenic activity. In addition,histology revealed more evident new cementum formation in theEGCG14-CS-Lovastatin1 group compared with that in Biomend®

and control groups. Although histomorphometry provides high res-olution and direct representations of alveolar bone levels, thereare obvious limitations such as tissue sample destruction andchallenges in three-dimensional image reconstruction (Park et al.,2007). Micro-CT provides significant advantages for identificationof mineralized structures with high accuracy. The new bone for-mation in the EGCG14-CS-Lovastatin1 group was superior basedon three-dimensional micro-CT analysis compared with that inBiomend® and control groups. Human randomized clinical trialsare required in the future to examine the clinical efficacy of theEGCG14-CS-Lovastatin1 membrane in periodontal tissue regenera-tion.

5. Conclusion

In this study, EGCG was grafted onto the surfaces of CS mem-branes, and lovastatin was included for controlled release throughthe membranes to confer multi-functionality. The EGCG14-CS-Lovastatin1 membrane showed good biocompatibility, effectiveantibacterial activity, increased ALPase activity, and significantlyincreased new bone formation in one-walled defects of a beagledog model. This membrane has the potential to be applied as anovel GTR membrane in periodontal regeneration surgery.

Conflicts of interest

The authors declare that they have no conflicts of interest.

Acknowledgements

This study was supported by grants (103-2314-B-002-093-MY3 and 103-2314-B-002-109) from the Ministry of Science andTechnology of Taiwan and grants (103-N2580, 104-S2626 and 104-N2846) from National Taiwan University Hospital.

Appendix A. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at http://dx.doi.org/10.1016/j.carbpol.2016.06.026.

http://dx.doi.org/10.1016/j.carbpol.2016.06.026http://dx.doi.org/10.1016/j.carbpol.2016.06.026http://dx.doi.org/10.1016/j.carbpol.2016.06.026http://dx.doi.org/10.1016/j.carbpol.2016.06.026http://dx.doi.org/10.1016/j.carbpol.2016.06.026http://dx.doi.org/10.1016/j.carbpol.2016.06.026http://dx.doi.org/10.1016/j.carbpol.2016.06.026http://dx.doi.org/10.1016/j.carbpol.2016.06.026http://dx.doi.org/10.1016/j.carbpol.2016.06.026http://dx.doi.org/10.1016/j.carbpol.2016.06.026http://dx.doi.org/10.1016/j.carbpol.2016.06.026

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