Wool Keratin Composite Membranes Support

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Bilayered PLGA/Wool Keratin Composite Membranes Support Periodontal Regeneration in Beagle Dogs Hualin Zhang, Juan Wang, Hairong Ma, Yueli Zhou, Xuerong Ma, Jinsong Liu, Jin Huang, and Na Yu ACS Biomater. Sci. Eng., Just Accepted Manuscript • DOI: 10.1021/acsbiomaterials.6b00357 • Publication Date (Web): 04 Oct 2016 Downloaded from http://pubs.acs.org on October 18, 2016

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ACS Biomaterials Science & Engineering

Bilayered PLGA/Wool Keratin Composite Membranes Support Periodontal Regeneration in Beagle Dogs Hualin Zhang,†,1,* Juan Wang,†,1 Hairong Ma,†,1 Yueli Zhou,† Xuerong Ma,† Jinsong Liu,‡,* Jin Huang,† and Na Yu† †

College of Stomatology, Ningxia Medical University, Yinchuan 750004, China

‡

School and Hospital of Stomatology, Wenzhou Medical University, Wenzhou

325027, China

AUTHOR INFORMATION

Corresponding Authors.

* Tel.: +86 15109519371. E-mail addresses: [email protected] (H.L. Zhang). * E-mail addresses: [email protected] (J.S. Liu). 1

These authors contributed equally to this work and should be considered co-first

authors.

FULL MAILING ADDRESS †

Shengli South Road #1160, Xingqing District, Yinchuan 750004, China

‡

College West Road #113, Lucheng District, Wenzhou 325027, China

ACS Paragon Plus Environment


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ABSTRACT: Regenerative procedures using guided tissue regeneration (GTR) membrane are presently well established in periodontal therapy. In this study, bilayered poly(lactic-co-glycolic acid) (PLGA)/wool keratin (WK) composite membranes were prepared for use as GTR membrane by the solvent casting and electrospinning methods. Then, the composite membranes were used to evaluate the effects in guided tissue regeneration in beagle dogs. The results showed that the bottom layer (casting film) of the bilayered PLGA/wool keratin composite membranes was a compact PLGA/wool keratin membrane, and the upper layer (electrospun film) was a loose, porous, three-dimensional, and fibrous PLGA/wool keratin membrane (The wool keratin was at five different levels: 0 wt%, 0.5 wt%, 1 wt%, 2 wt% or 4 wt%). The mechanical properties of the composites were significantly enhanced by the addition of wool keratin. The bilayered PLGA/1.0% wool keratin composite membranes presented the best values of ultimate strength and Young’s modulus. All of the bilayered PLGA/wool keratin composite membranes showed high thermal and thermooxidative stabilities. The GTR results showed that the PLGA/1.0% wool keratin composite membranes could effectively promote the periodontal tissue


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regeneration after 12 weeks, and have a similar effect to the collagen membrane on the regeneration of the periodontal tissue. Thus, the bilayered PLGA/wool keratin composite membranes show great potential to meet the demand for GTR membrane. The study will serve as a foundation for further study of the PLGA/wool keratin membranes for GTR application and the therapy of periodontal disease. KEYWORDS: PLGA, wool keratin, GTR membrane, electrospinning, periodontal disease, periodontal tissue regeneration

1. Introduction Periodontal disease, a chronic inflammatory condition affecting a large portion of the adult population in the world, is one of the major causes of tooth loss and it is also a major oral disease endangering human oral and systemic health.1,2 Periodontal disease is caused by a mixed infection of various periodontal pathogens (mainly anaerobic bacteria) which results in inflammation of the gums, leading to the gradual destruction of periodontal tissues and alveolar bone supporting the teeth.3,4 When the molar furcation area is affected by the periodontitis, the tissues in the corresponding region will be destroyed and the furcation involvement occurs. According to reports in the literature, the incidence of the furcation involvement was 26%-90%.5 Once the damage of the furcation area is formed, it is very difficult to remove the plaque and tartar in this area, and thus the periodontal disease will be accelerated or getting worse.



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At present, the treatment for furcation involvement includes periodontal flap surgery, bone grafting, periodontal guided tissue regeneration (GTR) and a series of methods based on GTR. Among them, GTR has been widely used to regenerate the lost periodontal tissue.6−13 In this treatment technology, GTR membrane is the key factor that directly affects the final regeneration results. Therefore, it is one of the urgent and difficult issues in this research field to find suitable barrier membrane material. The function of the biodegradable GTR membrane closely depends on the type, quality and design of the membrane materials. An ideal GTR membrane should be biocompatible and can biodegrade to nontoxic products within a specific time scale. It should be easy to be fabricated into proper mechanical properties and present a penetrating structure that plays a role of blocking the migration of gingival connective tissue cells and epithelial cells into the bone defect area as well as transporting both the nutrients and metabolic waste of the tissue.14

Single material or single structure is generally unable to fulfil the above requirements simultaneously, so researchers have focused on creating composite membranes made from combination of different types of materials with graded structure.15 For example, Liao16 developed a three-layered graded membrane with one face

of

nano-carbonated

hydroxyapatite/collagen/poly(lactic-co-glycolic

acid)

(nCHAC/PLGA) porous membrane, the opposite face of pure PLGA non-porous membrane, and the middle layer of nCHAC/PLGA porous membrane. This composite membrane, combining the biocompatibility of collagen, the osteoconductivity of


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HA, and the mechanical properties of PLGA, effectively avoids the aseptic inflammation caused by the degradation of PLGA while giving full play to the function of guided tissue regeneration. Similarly, Li17 developed a bilayered antibacterial GBR membrane that showed an asymmetric structure, in which the serosal layer fibres consisted of densely arranged collagen fibrils, whereas the external layer fibres consisted of irregularly aligned collagen fibrils. They concluded that the bilayered composite GBR membrane may provide a potential base to develop other bioactive GBR membranes. The above mentioned functional graded material points a new direction of the research on the GTR membrane: several single materials are put together to prepare a composite membrane of multi-layer structure by specific methods. PLGA and wool keratin are two excellent medical biomaterials that we have studied for a long time in our research group.14,18-22 These two materials have potential applications in the field of GTR.23-26 However, due to the lack of natural molecular recognition sites on PLGA surface, they can inhibit certain cell adhesion, limiting the application of PLGA in the biomedical field to a certain extent.27,28 Therefore, materials with good cell affinity, such as collagen, silk fibroin and hydroxyapatite, are often compounded with PLGA to improve the compatibility.29-31 Wool keratin, as a natural protein biomaterial, has been used to substitute for collagen so far.32-34 It can biodegrade to nontoxic products both in vitro and in vivo without causing inflammatory and immune responses.34 Moreover, it has better processing performance and can be processed into fibers, films, sponge structures, microcapsules,



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and so on.35-37 However, keratin or keratin-based materials have relatively poor mechanical properties. For example, pure wool keratin membrane is brittle.38 Therefore, we combine PLGA with wool keratin to prepare composite GTR membranes. On the one hand, wool keratin can improve the cell affinity and bioactivity of PLGA and reduce the incidence of PLGA-induced aseptic inflammation. On the other hand, PLGA can improve the mechanical properties of wool keratin, and thus make the composites accord with the requirements of ideal GTR membrane. In this study, a bilayered graded membrane, with the upper layer of PLGA/wool keratin porous membrane and the bottom layer of PLGA/ wool keratin non-porous membrane, was prepared by the methods of electrospinning and solvent casting, respectively. The dense layer can prevent the gingival epithelium and connective tissue from contacting the root surface and the porous layer can guide the periodontal ligament cells to grow along the root surface in the healing process; reform cementum, alveolar bone and periodontal ligament; establish the new attachment; and finally achieve periodontal tissue regeneration. Scanning electron microscope (SEM), Fourier transform infrared spectroscopy (FTIR), thermogravimetric analysis (TGA) and mechanical testing were used for characterization of the membranes. Then, the composite membranes were used in GTR surgery on beagle dogs. The study will serve as a foundation for further study of the bilayered PLGA/wool keratin membranes for GTR application and the therapy of periodontal disease.

2. Materials and methods


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2.1 Materials PLGA (Mw: 5×104 g/mol), with a lactide/glycolide ratio of 75:25 w/w, was purchased from Shandong Daigang Co., Ltd (Jinan, China). The wool was purchased from Kunshan Sanli Wool Carbonization Co., Ltd (Jiangsu, China). The wool keratin was extracted from clean wool. In brief, keratin was extracted by incubation in an aqueous solution of urea, mercaptoethanol, and sodium dodecyl sulfate at 60℃ for 5 h. The solution containing wool keratin was then dialyzed for 4 days and spray-dried to obtain WK powders. Dimethyl formamide (DMF), trichloromethane (TCM), and other analytical reagents were purchased from Tianjin Chemical Reagent Co., Ltd (Tianjin, China). Pentobarbital sodium was provided by Ningxia Medical University Affiliated Hospital. Primacaine was from Produits Dentaires Pierre Rolland (Merignac Cedex, France). Saline solution was from Shijiazhuang Siyao Co., LTD (Hebei, China). Hydrogen peroxide solution was from Texas Nu-lax Disinfection Products Co., LTD (Shandong, China). Chlorhexidine was from Jiangsu Morning Brand Bond Pharmaceutica Co., LTD (Jiangsu, China). Cefoperazone sodium and sulbactam sodium (250 mg/ml, 1 g/branch) were from Hunan Koren Pharmaceutical Co., LTD (Hunan, China). Collagen membrane (Lyoplant®, size: 1.5cm×3cm) was purchased from B/Braun (Melsungen, Germany). Lyoplant is a porous network of collagen fibres originating from bovine pericardium. Compont medical glue was from Beijing Compont Medical Glue Co., LTD (Beijing, China). Formalin was from Beijing HLCC Fine Chemicals Co., LTD (Beijing, China).

2.2. Bilayered composite membranes fabrication



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Medical grade PLGA containing wool keratin (1% wt) was utilized to fabricate the bottom layer of bilayered composite membranes via solvent casting method. The upper layer was prepared by electrospinning using an in-house solution spinning device.14 A 15-wt% PLGA solution was prepared by dissolving PLGA in a mixture of trichloromethane (TCM) and Dimethyl formamide (DMF) (7:3 v/v). The wool keratin (at five different levels: 0 wt%, 0.5 wt%, 1 wt%, 2 wt% or 4 wt%) was added to the PLGA solution. The solutions were sonicated for 1 h to accelerate the homogeneous dispersion of wool keratin. The blend solutions were loaded into a 5-mL plastic syringe with a blunt 18-gauge needle attached, and electrospun at a feed rate of 0.3 mL/min, at 15 kV and at a 16 cm tip to collector distance for 60 min. The syringe pump was connected to a moving device that could move in both directions equidistantly along a straight line that is through the center. The speed was set at 30 cm/min and amplitude at 2 cm, respectively. The bottom layer membrane (PLGA/wool keratin casting membrane) (10×10 cm2) was used to collect the electrospun fibers. After electrospinning, the bilayered PLGA/wool keratin (WK) membranes could be harvested. All procedures should carried out at room temperature.

2.3. Characterization of bilayered PLGA/WK composite membranes

2.3.1. Morphology The surface morphology of the membranes was observed by scanning electron microscopy (SEM; JSM-5900LV, JEOL, Tokyo, Japan) at 20 kV. The fiber diameter


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was determined by SEM by analyzing 50 single fibers using the Sigma Scan Pro 2.0 software (Systat Software, Inc., Chicago, IL, USA).

2.3.2. Chemical, thermal, and mechanical analyses FTIR spectra of the membranes were obtained using a Nicolet 560 FTIR spectrophotometer (Nicolet Instrument Co., WI, USA). The scan range was 4000 cm-1 －600 cm-1 with a resolution of 2 cm-1. The TGA thermograms were analyzed with a thermogravimetric analyzer (TAQ600; TA Instruments, New Castle, DE, USA) in a nitrogen atmosphere. The temperature was increased from room temperature to 400 °C at 10 °C/min. The weight of each specimen was maintained at ∼5 mg. Three samples were tested for each membrane type. Tensile testing was performed on the membranes using a universal testing machine (Instron 5567; TestResources, Inc., Shakopee, MN, USA) with a 100-N load cell at a crosshead speed of 5 mm/min at room temperature. All samples were cut into rectangles with dimensions of 1 cm×7 cm and the thickness of the sample was 100–150 µm. Six samples were tested for each membrane type.

2.4. Cell culture 100 µL (1×105 cells/mL) of the MC3T3 osteoblasts suspensions were seeded evenly into the bilayered PLGA and PLGA/1.0% WK composite membranes (1.0cm×1.0cm), respectively. The cell/membrane constructs were cultured in an incubator at 37℃ under humidified 5% CO2 conditions, and the cell culture medium was changed every 2 days. After 3 days of culture, the cell/membrane constructs were



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fixed with 2.5% glutaraldehyde in phosphate-buffered saline (PBS) (pH=7.3) at 4°C for at least 4 h. The samples were then dehydrated in a series of graded ethanol, followed by vacuum drying. The samples were sputter-coated with a thin layer of gold and

analyzed

by

SEM.

Cell

viability

was

measured

by

the

MTT

[3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrasodium bromide] assay. After 3 and 7 days of culture, 50 µL of MTT solution (5 mg/mL, Sigma) was added to each sample at 37°C for 4 h. The insoluble reaction product was then solubilized with 0.5 mL of dimethyl sulfoxide (DMSO). The absorbance was measured at 570 nm using a Microplate Reader (Benchmark, Bio-Rad, CA, USA).

2.5. In vivo experimental work Six healthy male periodontal disease-free beagle dogs (1-1.5 years, 10-15 kg) were selected, which were provided by the Kangping Experimental Animal Center (Shenyang, China, license key: SCXK (Liao) 2014-0003, quality certification number: 0001269-87-88). All in vivo experiments were approved by the Animal Ethics Committee of Ningxia Medical University. Bilateral mandibular second, third and fourth premolars were chosen as experimental teeth. The experiment was carried out after one week of adaptive breeding. All surgical procedures were performed under general anesthesia, achieved by an intraperitoneal injection of pentobarbital sodium (3%) at 0.5 ml/kg. Local infiltration of Primacaine was administered for hemostasis and the reduction of operative pain.

2.5.1. Periodontal measurement


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Three sites (mesial, intermediate and distal) on the buccal side of each experimental tooth should be examined prior to each of the procedures. All operations were performed by one operator. Pocket depth (PD): the distance between the bottom of periodontal pocket and the gingival margin. Attachment loss (AL): the distance between the bottom of periodontal pocket and the cemento-enamel junction (CEJ). Gingival recession (GR): the distance between the gingival margin and the CEJ. Bleeding on probing (BI): Mazza standards (1981).

2.5.2. Radiograph Standardized periapical radiographs were taken (DR3000, Kodak, China) prior to each of the procedures for a reference image of the furcation area.

2.5.3. Periodontal disease induction A week before induction, scaling was performed on all the experimental teeth with Gracey scalers (Shanghai Cambridge Dental Instrument Factory, Shanghai, China) to remove supragingival, subgingival calculus and soft dirt. Then, the teeth were rinsed with 0.9% saline solution and 3% hydrogen peroxide solution alternately. After administration of adequate local anesthesia, intrasulcular incisions were placed on the buccal and oral aspects of the experimental site, extending from the



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second premolar mesially to the fourth premolar distally. Vertical releasing incisions were placed for adequate access to the experimental site. Full-thickness flaps were then elevated to expose the buccal bone walls (Figure 1a and 1b). Class II furcation defects (5 mm apicocoronally (anatomical reference: CEJ at the center of the furcation), 5 mm mesiodistally and 2 mm buccolingually) (Figure 1b) were surgically created by high-speed turbine dental drill (TC-169, General Tooth Material Electronics Co., LTD, Henan, China) on the buccal side of the second (P2), third (P3) and fourth (P4) premolars. Periodontal ligaments and remnants on the root surfaces were removed by scaling, then the exposed defects were rinsed with 0.9% saline solution and 0.12% chlorhexidine alternately, and then the defects were completely filled with an impression material (3M, Dental products, Germany) (Figure 1c). The mucoperiosteal flaps were repositioned and then closed with a combination of interrupted and horizontal mattress sutures using non-resorbable 4-0 sutures (Weihai Grosvenor LTD's Medical Materials Co., LTD, Shandong, China) (Figure 1d). Cotton ball dipped in normal saline solution was used to press the wounds gently to eliminate the dead space. After surgery, cefoperazone sodium and sulbactam sodium were administered subcutaneously for 5 days to prevent infection. Dogs were fed with high sugar softened food to accelerate plaque accumulation. Sutures were removed after 10 days. The impression material was wiped off after 3 weeks. After that, plaque control was performed by scaling with Gracey scalers every week and the wounds were rinsed with 0.12% chlorhexidine every 24 h for 2 weeks. Five weeks after surgery, the periodontal disease induction was completed and the periodontal measurements and


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radiographs were performed then.

Figure 1. Class II furcation defects induction. (a) Periodontal condition before GTR surgery. (b) Full-thickness flaps were elevated to expose the buccal bone walls. Boundary of Class II furcation defects: 5 mm apicocoronally (anatomical reference: CEJ at the center of the furcation), 5 mm mesiodistally and 2 mm buccolingually. (c) The defects were completely filled with an impression material. (d) The wounds were repositioned and closed with 4-0 sutures.

2.5.4. Guided tissue regeneration Six beagle dogs were randomly and equally distributed into 6 weeks group and 12 weeks group. The three experimental teeth (P2, P3 and P4) on one side of each dog were divided further into three treatment groups: PLGA/1.0%WK composite membranes group (Group 1), collagen membrane group ( Group 2) and periodontal flap surgery group (Group3, control group). Thus, the sample size of each treatment group was six (n=6).



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A full-thickness flap was elevated once more to expose the defects. The root surfaces in the defects were all scaled and planned to remove the soft tissues, and then rinsed with sterile saline solution and 0.12% chlorhexidine solution (Figure 2a). Histologic reference notches on mesial and distal root surfaces in furcation area were made using 0.5mm ball drill (Figure 2b, blue arrow). The PLGA/WK composite membranes (Group 1, P3, Figure 2c, yellow arrow) and collagen membrane (Group 2, P2, Figure 2c, green arrow) were cut into appropriate sizes and smeared with a little compont medical glue on their edges, and then were immediately positioned to cover the defect, with the margins extending approximately 2-3 mm mesially, distally and apically (Figure 2c). The upper porous layer of the PLGA/WK composite membranes was facing the defect. The control group (Group 3, P4) had no membrane. Finally, the flap was closed with non-resorbable 4-0 sutures (Figure 2d). After surgery, cefoperazone sodium and sulbactam sodium were administered subcutaneously for 5 days to prevent infection. Soft diet was fed to avoid mechanical trauma. Sutures were removed 2 weeks after surgery. After that, plaque control was performed by scaling with Gracey scalers every week and surgical wounds were rinsed with 0.12% chlorhexidine every 24 h. Three dogs were euthanized by an overdose of general anesthetic at 6 weeks postoperatively and the remaining three at 12 weeks. The periodontal measurements and radiographs were performed for characterization.


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Figure 2. GTR surgery. (a) A full-thickness flap was elevated to expose the defects. (b) Histologic reference notches on mesial and distal root surfaces were made (0.5 mm depth, blue arrow). (c) PLGA/wool keratin composite membranes (yellow arrow) and collagen membrane (green arrow) were positioned to cover the defect with the margins extending approximately 2-3 mm mesially, distally and apically. (d) The wounds were repositioned and closed with 4-0 sutures.

2.6. CBCT The CBCT imaging system (NewTom VGi, QR S.R.L, Verona, Italy) was used to scan and reconstruct the specimens (layer thickness: 0.15 mm, scanning voltage: 120 KV, electricity: 5 mA and exposure time: 4 s). Indexes were measured as follows with measuring software: (1) Linear measurement: four consecutive section images in a buccolingual direction were selected to measure using NNT software in the region of interest (ROI) (accurate to 0.1 mm) (Figure 3a). Measurement indexes are as follows:



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Horizontal bone defect distance: the horizontal distance between CEJ and the deepest level of bone defect in furcation area. Vertical bone defect distance: the vertical distance between CEJ and the most coronal level of new alveolar bone in furcation area. Exposed root height (Figure 3b): the vertical distance between CEJ and the lowest point of the exposed root surface. New alveolar bone height (Figure 3c): the vertical distance between the bone defect edge and the most coronal level of new alveolar bone in furcation area. (2) Alveolar bone density measurement (Figure 3d): the obtained CBCT images were saved as DICOM format and input into Mimics 10.01 software (Materialis, Leuven, Belgium). Four consecutive images in a mesio-distal direction were selected in furcation area. Three oval ROI with an area of 0.5mm2 were chosen to measure. All the measurements were performed by the same person, measured once a week for three times in total, and the average value was taken as the final result.

Figure 3. Linear measurement. (a) The section images in buccolingual direction. (b) Exposed root height (red line). (c) Horizontal bone defect distance (yellow line), vertical bone defect distance (green line) and new alveolar bone height (red line). (d) Alveolar bone density measurement using Mimics 10.01 software in a mesio-distal direction in furcation area.


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2.7. Histology processing and analysis Bilateral mandibles of the dogs were intercepted (Figure 4a) and the experimental teeth and surrounding tissues in root furcation area were harvested (Figure 4b) and fixed in 10% formalin. Samples of each group at both time-points (6 and 12 weeks) were decalcified in 10% EDTA at pH 7.4 for 4 months and subsequently embedded in paraffin. The sections of 4 µm-thick, cut in a mesio-distal direction, were used for Hematoxylin-eosin (HE) and Masson staining. Morphological observation was performed using a microscope (DFC425, Leica, Germany) and an image analysis system (LAS Version 3.7.0, Leica, Germany). IPP 6.0 image analysis software (Image-Pro Plus, Media Cybernetics, Inc., USA) was used to calculate the following parameters:

(1) New cementum (NC): the distance between the bottom of apical notch and the top of new cementum. (2) New periodontal ligament (NP): the distance between the bottom of apical notch and the top of new periodontal ligament. (3) Total defect length (TDL): total length between CEJ and the bottom of apical notch. (4) New alveolar bone area (NBA): the area of newly formed alveolar bone in furcation area. (5) Total defect area (TDA): the area of the original defect in furcation area.



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Figure 4. (a) The mandible was intercepted. Silicon carbide slice was used to separate each experimental tooth according to the marked lines (yellow lines), and the enamel was removed (green line). M represents mesio and D represents distal. (b) The experimental teeth and surrounding tissues in root furcation area were harvested (4 mm-thick in buccolingual direction).

2.8. Statistical analysis Statistical package for social science 17.0 software (SPSS17.0, USA) was used to analyze the data. Means and standard deviations were calculated for each measured parameter. For the PD, AL and GR parameters, T test was used. For BI parameter, Rank sum test for two samples was used. For CBCT parameters and histological measurements, the data should be tested for homogeneity of variance and normal distribution. If the data satisfy the requirements of normal distribution and homogeneity of variance, One-way ANOVA was used among the groups, and least-significant difference (LSD) was used for further comparison between the two groups. Two independent-sample T test was used for comparison in the same group. If not, K independent sample nonparametric tests and two independent sample


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nonparametric tests were used; p< 0.05 was considered as statistically significant.

3. Results and discussion

3.1. Morphology of the bilayered PLGA/WK membranes The fabrication technique of the bilayered PLGA/WK composite membranes and photographs of the PLGA/WK casting film and bilayered composite membranes are shown in Figure 5. The upper layer of the bilayered composite membranes was a electrospun PLGA/WK film and the bottom layer was a PLGA/WK casting film (Figure 5c).

Figure 5. Fabrication scheme of the bilayered composite membranes. (a) Photograph of PLGA/WK casting film. (b) Electrospinning process. The PLGA/WK casting film was used to collect the electrospun fibers. (c) Model diagram and (d) photograph of the bilayered PLGA/WK composite membranes. Five groups of PLGA/WK composite membranes with double-layer structure were prepared by the solvent casting method (bottom layer) and the electrospinning method (upper layer). Figure 6 shows SEM images of the bilayered composite membranes and Table 1 shows the corresponding average diameters of the electrospun fibers (upper layer) with various concentrations of wool keratin (ranging from 0 to 4.0



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wt%). The bottom layer was a PLGA/WK membrane with dense structure (Figure 6a and 6b) and the upper layer was a loose and porous PLGA/WK membrane with three-dimensional fibrous structure (Figure 6c, 6e, 6g, 6i and 6k). The bilayered structure of the composite membranes can be clearly seen by the SEM cross-sectional views (Figure 6d, 6f, 6h, 6j and 6l). The electrospun PLGA/WK fibers were smooth, uniform and continuous, suggesting that the wool keratin was effectively wrapped in the PLGA matrix. The average fiber diameters of the bilayered PLGA, PLGA/0.5% WK, PLGA/1% WK, PLGA/2% WK and PLGA/4% WK composite membranes were analyzed to be 288±67 nm, 430±81 nm, 342±86 nm, 216±54 nm and 470±92 nm respectively, using Sigma Scan Pro 2.0 software.

Figure 6. SEM images of the bilayered composite membranes. (a) The PLGA/WK casting membrane. (b) Cross-sectional view of a. (c) The bilayered PLGA composite membranes. (d) Cross-sectional view of c. (e) The bilayered PLGA/0.5% WK


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composite membranes. (f) Cross-sectional view of e. (g) The bilayered PLGA/1.0% WK composite membranes. (h) Cross-sectional view of g. (i) The bilayered PLGA/2.0% WK composite membranes. (j) Cross-sectional view of i. (k) The bilayered PLGA/4.0% WK composite membranes. (l) Cross-sectional view of k.

Table 1. The average diameters of the electrospun fibers

Sample

Average diameter

(Bilayered electrospun fiber)

(nm)

PLGA fiber

288±67

PLGA/0.5% WK fiber

430±81

PLGA/1.0% WK fiber

342±86

PLGA/2.0% WK fiber

216±54

PLGA/4.0% WK fiber

470±92

3.2. Characterization of the bilayered PLGA/WK membranes The FTIR spectra of the PLGA/WK casting film, bilayered PLGA, PLGA/0.5% WK, PLGA/1.0% WK, PLGA/2.0% WK, and PLGA/4.0% WK composite membranes and WK powders are shown in Figure 7a. The strong broad peak of the WK powders at 3300–3500 cm−1 is characteristic of the N–H stretching, and the peaks at 1650, 1534, and 1230 cm−1 are characteristic of the C=O stretching for amide I, N– H bending and C–H stretching for amide II, and C–N stretching and N–H bending for amide III, respectively. The intense peak at 1023 cm−1 indicates the S–O symmetric stretching vibrations of cysteine-S-sulfonate residues. In addition, the absorption at 1650, 1630–1520, and 1230 cm−1 suggests the presence of α-helices, β-sheets, and



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random coil structures, respectively.22 For the spectra of the bilayered PLGA membrane, wavenumbers from 1049 to 1130 cm-1 are the C–CH3 stretching vibrations. The peaks at 1750 cm-1, 1451cm-1, and 1186 cm-1 correspond to the C=O stretching, C–H bending and C–O stretching vibrations of PLGA, respectively. The spectra of the four bilayered PLGA/WK membranes and PLGA/WK casting film were very similar to that of the bilayered PLGA membrane, but after careful observation, we found that FTIR spectra of the bilayered PLGA membrane have no peaks from 1700 to 1500 cm-1. For PLGA/0.5% WK and PLGA/2.0% WK composite membranes, one peak have appeared at 1600-1700 cm-1 which belongs to keratin. For PLGA/4.0% WK composite membranes, two peaks have appeared at 1600-1700 cm-1 and 1550 cm-1 which also belong to keratin. These peaks were very weak and this may be due to that the wool keratin particles were well dispersed within the fibrous membranes, not just appearing on the surface. The mechanical properties of the bilayered PLGA and PLGA/WK membranes are summarized in Table 2. The tensile stress–strain curves of the bilayered PLGA and PLGA/WK membranes are shown in Figure 7b. The tensile strength and the elongation at break of the bilayered PLGA composite membranes were 5.45 MPa and 78.30 %, respectively. Compared to the bilayered PLGA composite membranes, the tensile strength (from 5.45 to 5.68 MPa) and Young’s modulus (from 338.74 to 341.72.61 MPa) of the bilayered PLGA/0.5%WK composite membranes increased slightly, but the elongation at break decreased (from 78.30 to 51.90%). When the wool keratin increased from 0.5 % to 1.0 % , the tensile strength and Young's modulus of


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the composite membranes increased significantly (the tensile strength increased by 63%, and the Young’s modulus increased by 73%), but the elongation at break decreased slightly, suggesting that the bilayered PLGA/1.0% WK membranes were tougher and more resistant to deformation. When the wool keratin content continued to increase to 2% and 4%, the tensile strength and Young's modulus of the composite membranes were smaller than those of the PLGA/1.0% WK membranes but were larger than those of the bilayered PLGA membranes. This indicated that the mechanical properties of the composite membranes were significantly increased after the addition of the wool keratin, and that even a small amount of wool keratin could significantly improve the tensile properties of the composite membranes. This enhancement may be due to the interaction between the homogeneously dispersed wool keratin and the polymer chains. Thermal analysis was used to detect the thermal stability of the bilayered composite membranes. All of the composite membranes underwent one step decomposition. The initial decomposition temperatures at weight losses of 10 %, 20 %, 60 %, and 90 % (T10 %, T20 %, T60 %, and T90 % values, respectively) of the four bilayered PLGA/WK composite membranes were all higher than those of the bilayered PLGA membranes by 10–30 °C (Table 3), indicating a higher thermal stability and thermal oxidative stability. It is also proved that even a small amount of wool keratin can greatly increase the thermal stability of the polymer matrix. The maximum thermal degradation temperatures that were determined from the first-derivative peak in Figure 7c were 338.77℃, 348.82℃, 356.27℃, 354.03℃, and



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358.77℃, respectively (Figure 7d), and the degradation temperature increased as the wool keratin content increases. The bilayered PLGA/WK membranes were thermally more stable than the bilayered PLGA membranes, consistent with our previous findings. This may be due to the higher crystallinity of the β-sheet structure, where the intermolecular interactions among the protein chains are stronger.21 Moreover, PLGA interferes with the keratin self-assembling causing the protein to assume a secondary structure thermally more stable.39 Thermal analysis pointed out that the polymer and wook keratin interfere each other in the supramolecular arrangements.39

Figure 7. (a) The FTIR spectra of the (A) PLGA/WK casting film, bilayered (B) PLGA, (B) PLGA/0.5% WK, (C) PLGA/1% WK, (D) PLGA/2% WK and (E) PLGA/4% WK composite membranes, and (G) WK powders. (b) The tensile stress– strain curves of the bilayered PLGA, PLGA/0.5% WK, PLGA/1% WK, PLGA/2%


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WK and PLGA/4% WK composite membranes. (c) TGA results of the bilayered PLGA, PLGA/0.5% WK, PLGA/1% WK, PLGA/2% WK and PLGA/4% WK composite membranes: thermogravimetric traces and (d) first derivative of the curves (a). Table 2. The mechanical properties of the bilayered PLGA, PLGA/0.5% WK, PLGA/1% WK, PLGA/2% WK and PLGA/4% WK composite membranes.

Sample

Tensile Strength

Elongation at break

(MPa)

(%)

Young's modulus (MPa)

PLGA/WK casting membrane

9.77

40.20

651.40

Bilayered PLGA membrane

5.45

78.30

338.74

Bilayered PLGA/0.5%WK membrane 5.68

51.90

341.72

Bilayered PLGA/1.0%WK membrane 9.26

48.60

590.77

Bilayered PLGA//2.0%WK membrane 5.48

54.60

393.88

Bilayered PLGA//4.0%WK membrane 6.57

75.10

424.94

Table 3. Thermal stabilities of the bilayered PLGA, PLGA/0.5% WK, PLGA/1% WK, PLGA/2% WK and PLGA/4% WK composite membranes.



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Sample

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T10%

T20%

T60%

T90%

(℃)

(℃)

(℃)

(℃)

PLGA membrane

268.24

291.74

313.56

335.30

PLGA/0.5%WK membrane

250.18

289.26

328.87

346.53


281.62

305.84

342.45

362.03


277.49

306.47

347.22

365.81


275.03

305.17

348.47

367.28

3.3. Cell culture The morphologies of MC3T3 cells on the bilayered PLGA and PLGA/1.0% WK composite membranes are shown in Figure 8a and 8b. After 3 days of culture, more MC3T3 osteoblasts adhered on the surface of the bilayered PLGA/1.0% WK composite membranes than on the bilayered PLGA membranes, indicating that the PLGA/1.0% WK composite membranes had a high affinity for MC3T3 osteoblasts and supported the attachment and growth of cells more efficiently. Figure 8c shows the MTT assay of MC3T3 osteoblasts seeded on the bilayered PLGA and PLGA/1.0% WK composite membranes after 3 and 7 days of culture. There were significantly more MC3T3 osteoblasts on the PLGA/1.0% WK membranes both at 3 and 7 days than on the PLGA control (p < 0.05). Cell culture results suggested that after the addtion of wool keratin, the bilayered PLGA/1.0% WK composite membranes more tended to promote attachment and proliferation of MC3T3 osteoblasts compared to


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the bilayered PLGA membranes.

Figure 8. SEM images of MC3T3 osteoblasts cultured on (a) bilayered PLGA and (b) PLGA/1.0% WK composite membranes after 3 days of culture. More cells were observed on the bilayered PLGA/1.0% WK composite membranes than on the bilayered PLGA membranes. (c) MTT assay of MC3T3 osteoblasts cultured on the bilayered PLGA and PLGA/1.0% WK composite membranes at 3 and 7 days. Error bars represent means±SD, n=3, ‘*’ represents p < 0.05.

3.4. Periodontal disease induction The periodontal tissue of the beagle dogs had no gingival congestion, swelling and alveolar bone defect before the induction of periodontal disease. The silicone rubber was wiped off after 3 weeks (Figure 9a). After 5 weeks of induction, soft dirt was



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found on the surface of the experimental teeth; the receding gums were red, swelling and bled easily on probing; and the depth of periodontal pocket was significantly increased (Figure 9b), all suggesting that the periodontal tissues were in an obvious inflammatory state. X-ray images showed that the density of alveolar bone in furcation area reduced after periodontal disease induction (Figure 9c and 9d). Before induction, the PD was less than 1 mm; after induction, the PD increased to between 1 mm and 2 mm, a statistically significant growth (P0.05). This may be due to the short observation time after GTR surgery and small new alveolar bone mass. The CBCT results showed that the alveolar bone regeneration effect of Group 1 and Group 2 surpassed that of Group 3 both at 6 weeks and 12 weeks and the alveolar bone regeneration effect of the first two groups at 12 weeks were stronger than that at 6 weeks; there was less alveolar bone regeneration in Group 3 and there was no significant change between 6 and 12 weeks. It is hard for X-ray film to accurately reflect the real situation of local bone defects due to the projection angle, position of film, artificial operation error and so on, and thus there was some difference between the X-ray images and the clinical pathological changes, and the result is not accurate. Therefore, X-ray examination can only be used as an auxiliary examination for clinical diagnosis. In this study, three-dimensional CBCT was used in order to make an accurate judgment on the


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situation of the new alveolar bone in the root furcation area. In recent years, CBCT has been widely used in oral clinical diagnosis due to its short acquisition time, high accuracy and image resolution, rapid scanning speed, low radiation dose, cheap price and so on.40-42 CBCT images can accurately display the defect of alveolar bone at different levels and it can also be used to evaluate the status of alveolar bone in the therapy of periodontal disease. Many researchers have done further measurement and analysis with the relevant measurement software.43,44 In this study, NNT software and Mimics 10.01 software were used to analyze the results obtained from CBCT examination. So far, similar report of combining the use of CBCT and the relevant software for the measurement of alveolar bone is relatively scarce. Therefore, the accuracy of alveolar bone density and linear measurement still needs further study. Table 8. CBCT measurements after GTR surgery (n=6, x±s)

Parameter

Group

6 weeks

ABD

1

930.3417±23.82△

1154.26±103.22△*

(Hu)

2

901.52 ±40.14☆

1051.34±29.68☆♯

3

701.36±177.73

849.22 ±77.76

ABH

1

1.54±0.35△

1.68±0.08△

(mm)

2

1.50±0.19☆

1.57±0.19☆

3

0.98±0.41


12 weeks

1.17±0.14


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REL

1

4.06±0.25△

3.95±0.18△

(mm)

2

4.17±0.40☆

4.09±0.2☆

3

4.51±0.13

4.48±0.34

HBDD

1

1.64±0.44

1.82±0.08

(mm)

2

1.99±0.38

1.86±0.18

3

1.99±0.46

1.87±0.28

VBDD

1

4.18±0.36

4.26±0.16

(mm)

2

4.12±0.71

4.15±0.42

3

4.53±0.20

4.41±0.37

△

Significant difference between Group 1 and Group 2 (P﹤0.05)

☆


* Significant difference between 6 weeks and 12 weeks in Group 1 (P﹤0.05) ♯

Significant difference between 6 weeks and 12 weeks in Group 2 (P﹤0.05)

3.7. Histological analysis

3.7.1. Six weeks after GTR surgery Bilayered PLGA/WK membranes group (Figure 11a-c): part of the fibrous connective tissue and alveolar bone with macrovoid above the notch could be seen in the furcation area under light microscope. There were many new cellular cementum and new periodontal ligament in local area. Masson staining showed that the maturity of the new alveolar bone, mainly blue dyed, was lower than that of normal bone.


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Collagen membrane group (Figure 11d-f): much fibrous connective tissue and a small amount of inflammatory cell infiltration in local area could be seen in the furcation area. New alveolar bone with loose structure above the notch revealed low maturity. Long new cementum with mixed type could be seen around the maker and preliminary formed periodontal ligament could be found in local area. Masson staining showed blue dominated new alveolar bone and blue stained collagen fibers, suggesting that the calcification and maturity of the new alveolar bone were both low. Periodontal flap surgery group (Figure 11g-i): a large amount of fibrous connective tissue and inflammatory cells were found in the furcation area. A few newly formed alveolar bone and cellular cementum were formed around the notch, and new periodontal ligament was unapparent, showing that the periodontal repair was mainly formed by the formation of long junctional epithelial, and that the regeneration of periodontal tissue was minimal. New alveolar bone with low maturity was mainly dyed blue in Masson staining.



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Figure 11. Representative histological sections of samples stained with H&E and Masson at 6 weeks. DE: dentin, NB: new alveolar bone, NC: new cementum, and PDL: periodontal ligament. (a) Fibrous connective tissue (yellow arrow) and new alveolar bone in furcation area (4×, HE). (b) Higher magnification of the green selected area in Figure 11a. Capillaries in the newly formed periodontal ligament (yellow arrow) (20×, HE). (c) Higher magnification of the red selected area in Figure 11a. Mainly blue dyed new alveolar bone (20×, Masson). (d) Fibrous connective tissue (yellow arrow) and newly formed alveolar bone above the notch (4×, HE). (e) Higher magnification of the green selected area in Figure 11d. Capillaries in the newly formed alveolar bone (green arrow) and periodontal ligament (yellow arrow) (20×, HE). (f) Higher magnification of the red selected area in Figure 11d. Mainly


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blue dyed new alveolar bone (20×, Masson). (g) Fibrous connective tissue (yellow arrow) and a small amount of newly formed alveolar bone above the notch (4×, HE). (h) Higher magnification of the green selected area in Figure 11g. New periodontal ligament is not obvious (yellow arrow) (20×, HE). (i) Higher magnification of the red selected area in Figure 11g. Blue dyed new alveolar bone (20×, Masson).

3.7.2. Twelve weeks after GTR surgery Bilayered PLGA/WK membranes group (Figure 12a-c): less fibrous connective tissue was found under light microscope. Newly formed alveolar bone was in great quantities above the notch and a large amount of new cellular cementum was seen near the furcation area. A large number of collagen fiber bundles were embedded into the new alveolar bone and cementum in some regions. Masson staining showed that red and blue dyed mature alveolar bone and new alveolar bone intertwined together and red stained new alveolar bone was more mature. This group showed preferable periodontal regeneration effect with more new periodontal tissues formation. Collagen membrane group (Figure 12d-f): less fibrous connective tissue was found under light microscope and local lymphocyte infiltration was still visible. A large amount of new alveolar bones with obvious bone lacuna were seen above the maker. Periodontal ligament containing abundant capillaries and collagen fiber bundles were embedded into new alveolar bone and cementum in some regions. Masson staining showed that newly formed alveolar bone was dyed red and blue, indicating that the maturity of the new alveolar bone was increased.



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Periodontal flap surgery group (Figure 12g-i): a large amount of fibrous connective tissue, gingival epithelial and less new alveolar bone were found in the furcation area. Clear boundary could be seen between the new and old cementum, and newly formed periodontal ligament was not obvious. Masson staining showed immature blue dyed alveolar bone in this group, but the bone maturity was increased compared to that of 6 weeks. Masson staining has specificity to the ossein and the represented color is associated with the maturity of ossein. The bone tissue is blue dyed in the early stage, blue and red dyed in the mature process, and converted into red dyed after maturation. The more the proportion of red color is, the more mature the bone is. Because this staining method has the advantages of bright color, stable dyeing, sharp contrast, easy identification, etc., it is generally used to evaluate the maturity of bone. In this study, Masson staining results indicated that the maturity of the new alveolar bone with blue and red dyed in Group1 and Group 2 was better than that (blue dyed) in Group 3, and each groups at 12 weeks showed higher degree of calcification than 6 weeks, which was consistent with the results of the previous researches.45,46


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Figure 12. Representative histological sections of samples stained with H&E and Masson at 12 weeks. DE: dentin, NB: new alveolar bone, NC: new cementum, and PDL: periodontal ligament.(a) A lot of new alveolar bone and a small amount of fibrous connective tissue (yellow arrow) in furcation area (4×, HE). (b) Higher magnification of the green selected area in Figure 12a. (20×, HE). (c) Higher magnification of the red selected area in Figure 12a. Red dyed new alveolar bone (20×, Masson). (d) Fibrous connective tissue (yellow arrow) and newly formed alveolar bone (above the notch (4×, HE). (e) Higher magnification of the green selected area in Figure 12d. Rich capillaries in the newly formed periodontal ligament (yellow arrow) (20×, HE). (f) Higher magnification of the red selected area in Figure 12d. Blue and red dyed new alveolar bone (20×, Masson). (g) A large amount of fibrous connective tissue, gingival epithelial (yellow arrow) and less new alveolar



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bone in furcation area (4×, HE). (h) Higher magnification of the green selected area in Figure 12g. Clear boundary between new cementum and the old (yellow arrow), and collagen fiber bundles were not obvious (green arrow) (20×, HE). (i) Higher magnification of the red selected area in Figure 12g. Blue and red dyed new alveolar bone (green arrow) (20×, Masson). At 6 weeks after GTR surgery, the PLGA/WK composite membranes was not absorbed completely; but at 12 weeks, the residual film was not found in the defect area. This indicated that the absorption time of the bilayered PLGA/WK composite membranes was between 6 and 12 weeks, and this time period is the key stage of periodontal tissue repair and regeneration. In theory, the degradation rate of the membrane should be consistent with the rate of tissue regeneration in order to achieve the desired effect. If the degradation rate of the membrane is reduced, the membrane will have enough time to maintain the regeneration space and the amount of the regenerated periodontal tissue may increase. In this study, we found that the effect of periodontal tissue regeneration is still far less than the ideal regeneration effect, and the newly formed bone in the furcation area, which is mainly the lamellar bone, was not mature. However, further studies by extending the observation time and adjusting the molecular weight of PLGA in order to get better effect of periodontal tissue regeneration are needed. Melcher concluded that the healing method of the periodontal tissues (epithelial cells, connective tissue cells, periodontal ligament cells and bone cells) was determined by the initial growth of periodontal cells on the root surface.48 The barrier


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membrane can prevent epithelial cells from contacting with the root surface in the early stage, maintaining space for periodontal tissue regeneration. In our study, the regeneration effect was poor without the barrier membrane in Group 3 due to the premature contact of epithelial tissue and the root surface, and the defect was mainly filled by the connective tissue. Researches have indicated that it is rather difficult to achieve complete regeneration of periodontal bone defect with area larger than 4mm2.49 In this study, a blank area and a small amount of connective tissue were found at the top of the furcation area in the individual tooth, and the periodontal tissue regeneration still did not achieve the best effect. The exposure of GTR membrane is easy to cause the deposition of bacteria and the postoperative infection caused by bacteria is now considered the main cause of GTR failure.50 Therefore, effective measures should be taken to avoid the exposure of GTR membrane.

3.7.3. Quantitative analysis The results of quantitative analysis were shown in Table 9. At 6 weeks after GTR surgery, the percentage of New cementum (NC) of Group 1, Group 2 and Group 3 were 28.00±15.57 %, 29.33±5.09 % and 12.50±9.05 %, respectively. At 12 weeks, the NC% of the three groups were 32.83±9.52 %, 32.67±7.23 % and 17.17±9.06 %, respectively. The NC% in Group 1 and Group 2 were significantly higher than that in the control group and there was statistical difference between the treatment groups (Group 1 and Group 2) and the control (Group 3) at 6 weeks and 12 weeks (P0.05). Besides, there was no statistical difference in the same group between 6 weeks and 12



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weeks (P>0.05). As for the percentage of new periodontal membrane (NP) and new alveolar bone area (NBA), the results were similar to that of the NC%. There was statistical difference between the treatment groups (Group 1 and Group 2) and the control (Group 3) at 6 weeks and 12 weeks (P0.05). Besides, there was no statistical difference in the same group between 6 weeks and 12 weeks (P>0.05).

Table 9.

The NC%, NP% and NBA% ( n=6, %, x ± s)

Parameter NC%

NP%

NBA%

Group

6 weeks

12 weeks

1

28.00±15.57△

32.83±9.52△

2

29.33±5.09☆

32.67±7.23☆

3

12.50±9.05

17.17±9.06

1

26.67±6.22△

28.17±4.71△

2

21.50±13.72☆

24.00±7.92☆

3

8.50±7.58

12.00±4.52

1

19.00±7.69△

28.17±7.88△

2

17.50±7.58☆

24.67±4.63☆

3

7.33±6.35

14.67±13.17

△


☆


In a word, at 6 weeks after GTR surgery, a small quantity of new alveolar bone and cellular cementum could be seen in Group 1 and Group 2, and there was no collagen fiber bundles connecting the cementum and alveolar bone. At 12 weeks after


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GTR surgery, the amount of the new periodontal tissue was more than that of 6 weeks. A large number of new alveolar bones with plenty of capillaries in them were found in Group 1 and Group 2. Beside, collagen fibers bundles were inserted into the new cementum and new alveolar bone in some regions, indicating that better regeneration effect of periodontal tissues was achieved in these two groups. However, there were more fibrous connective tissue and less newly formed periodontal tissues in Group 3, indicating worse regeneration effect than Group 1 and Group 2. Moreover, the percentage of NC, NP and NBA in Group 1 and Group 2 were significantly higher than those in the control group. Masson staining results indicated that the maturity of the newly formed alveolar bone with blue and red dyed in Group 1 and Group 2 was higher than that (mainly blue dyed) in Group 3, and the calcification degree of the new alveolar bone in all the three groups at 12 weeks was higher than that at 6 weeks. Therefore, the bilayered PLGA/WK membranes and collagen membrane both had good periodontal regeneration ability and the PLGA/WK membranes had similar effect in prompting periodontal tissue regeneration as the collagen membrane.

4. Conclusion The bilayered composite membranes composed of PLGA and WK with different WK contents were fabricated using solvent casting and electrospinning methods. WK may increase the mechanical and thermal properties of the composites also at low concentrations. The bilayered PLGA/wool keratin composite membranes had a good effect on guiding the periodontal tissue regeneration, and the effect of regeneration was similar to that of the collagen membrane. Therefore, the bilayered PLGA/wool



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keratin composite membranes have a great potential as a new type of GTR membrane. Notes The authors declare no competing financial interest.

ACKNOWLEDGMENTS This work was supported by National Natural Science Foundation of China (No. 81200818, No. 81360269, No.81560191 and No. 81371182).

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TABLE OF CONTENTS GRAPHIC

Bilayered PLGA/Wool Keratin Composite Membranes Support Periodontal Regeneration in Beagle Dogs Hualin Zhang,†,1,* Juan Wang,†,1 Hairong Ma,†,1 Yueli Zhou,† Xuerong Ma,† Jinsong Liu,‡,* Jin Huang,† and Na Yu†



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TOC 199x86mm (300 x 300 DPI)


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Wool Keratin Composite Membranes Support

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