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Hierarchically Patterned PolydopamineContaining Membranes for Periodontal Tissue Engineering Downloaded via ALBRIGHT COLG on March 21, 2019 at 13:17:10 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.
Mohammad Mahdi Hasani-Sadrabadi,†,‡,§,∥ Patricia Sarrion,† Nako Nakatsuka,‡,§ Thomas D. Young,‡,§ Nika Taghdiri,†,△ Sahar Ansari,† Tara Aghaloo,⊥ Song Li,∥,# Ali Khademhosseini,‡,∥,#,¶ Paul S. Weiss,*,‡,§,#,□ and Alireza Moshaverinia*,†,‡,# †
Weintraub Center for Reconstructive Biotechnology, Division of Advanced Prosthodontics, School of Dentistry, University of California, Los Angeles, Los Angeles, California 90095-1668, United States ‡ California NanoSystems Institute, University of California, Los Angeles, 570 Westwood Plaza, Los Angeles, California 90095-7227, United States § Department of Chemistry and Biochemistry, University of California, Los Angeles, 607 Charles E. Young Drive South, Los Angeles, California 90095-1569, United States ∥ Department of Bioengineering, University of California, Los Angeles, 420 Westwood Plaza, 5121 Engineering V, Los Angeles, California 90095-1600, United States ⊥ Division of Diagnostic and Surgical Sciences, School of Dentistry, University of California, Los Angeles, Los Angeles, California 90095-1668, United States # Center for Minimally Invasive Therapeutics (C-MIT), University of California, Los Angeles, Los Angeles, California 90095-7227, United States ¶ Department of Chemical and Biomolecular Engineering, University of California, Los Angeles, Los Angeles, California 90095-1592, United States □ Department of Materials Science and Engineering, University of California, Los Angeles, 410 Westwood Plaza, Los Angeles, California 90095-1595, United States S Supporting Information *
ABSTRACT: Periodontitis is a common chronic inflammatory disease that affects tooth-supporting tissues. We engineer a multifunctional periodontal membrane for the guided tissue regeneration of lost periodontal tissues. The major drawback of current periodontal membranes is the lack of tissue regeneration properties. Here, a series of nanofibrous membranes based on poly(ε-caprolactone) with tunable biochemical and biophysical properties were developed for periodontal tissue regeneration. The engineered membranes were surface coated using biomimetic polydopamine to promote the adhesion of therapeutic proteins and cells. We demonstrate successful cellular localization on the surface of the engineered membrane by morphological patterning. Polydopamine accelerates osteogenic differentiation of dental-derived stem cells by promoting hydroxyapatite mineralization. Such multiscale designs can mimic the complex extracellular environment of periodontal tissue and serve as functional tissue constructs for periodontal regeneration. In a periodontal defect model in rats, our engineered periodontal membrane successfully promoted the regeneration of periodontal tissue and bone repair. Altogether, our data demonstrate that our biomimetic membranes have potential as protein/cell delivery platforms for periodontal tissue engineering. KEYWORDS: polydopamine coating, periodontal membrane, osteogenic differentiation, microscale patterning
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eriodontal disease is one of the most common chronic dental infections and is a destructive inflammatory disease.1 Periodontitis, the more serious form, can © XXXX American Chemical Society
Received: December 20, 2018 Accepted: March 11, 2019
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Figure 1. (a) Schematic of the electrospinning process used to make functional periodontal membranes. A solution of poly(ε-caprolactone) (PCL) (10% in HFIP) was electrospun at 15 kV with an infusion rate of 2.5 mL/h and a tip-to-target gap of 10 cm. (b) Schematic overview of the self-polymerization reaction implemented to coat a PCL membrane with polydopamine (PDA). (c) Scanning electron microscopy (SEM) images of prepared nanofibers (top) before and (bottom) after PDA deposition. (d) Morphological patterning of nanofibrous membrane achieved using metal mesh as fiber collectors. (e) Changing the filament diameter or mesh opening can control the final microstructure, as shown for two meshes. (f) Relationship between time of polymerization and deposition thickness as well as density of deposited PDA. The presented data are plotted as mean ± SD. Surface chemical composition of nanofibers as investigated by X-ray photoelectron spectra for (g) PCL and (h) PDA−PCL. High-resolution spectra of nitrogen peaks (N 1s) for PCL and PDA-coated PDA are presented on the right.
entiation into osteoblasts, fibroblasts, and cementoblasts.8,9 However, for high-quality periodontal tissue regeneration, periodontal membranes used in GTR should act not only as barriers but also as delivery vehicles to release drugs and/or bioactive molecules including antibiotics and growth factors.5,10 In addition, GTR membranes must have suitable physicomechanical properties (e.g., elastic modulus and degradation profile) enabling their application in periodontal defects. For example, for regeneration, the membrane must be physically stable in the defect site during healing.11 Currently, both nonabsorbable (e.g., polytetrafluoroethylene, PTFE) and absorbable (e.g., collagen or other polymeric) membranes are available for GTR.12 Studies have shown that nonabsorbable membranes can reduce pocket depth and improve attachment gain, and result in periodontal tissue regeneration.13,14 Unfortunately, a second surgery is required to remove the membrane, leading to crestal bone resorption and compromising the wound-healing process. Alternatively, absorbable membranes have been used to prevent these setbacks. However, absorbable membranes have other disadvantages, including unpredictable degradation rates, poor mechanical
result in the destruction of the periodontium, which is a set of specialized tissues that support the teeth, including alveolar bone, periodontal ligaments, and cementum.1,2 Left untreated, periodontitis results in progressive loss of periodontal attachment and surrounding bone loss, as well as early loss of teeth.3 Periodontal therapy targets the regeneration of the entire periodontium. Recent periodontal repair strategies have focused primarily on applying conventional antibiotics, guided tissue regeneration (GTR), and the administration of growth factors and other bioactive agents.4 However, systematic reviews of these procedures have shown inconsistent results and variable outcomes.5 Guided periodontal tissue regeneration strategies using periodontal membranes have been suggested.5−7 During healing, periodontal membranes serve as mechanical barriers that prevent or retard the migration of the junctional epithelium; they enable periodontal ligament and bone tissue to repopulate the root surface selectively. Typically, in GTR procedures, polymeric films/membranes are used to obtain an isolated, physically protected surgical niche capable of recruiting local progenitor cells and supporting their differB
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Figure 2. (a) Mechanical characterization of poly(ε-caprolactone)−polydopamine (PCL−PDA)-based electrospun membranes as tested by uniaxial tensile experiments. (b) Tensile strength (in MPa), elongation at break (in %), and Young’s modulus (in MPa) determined from force−displacement data (expressed as mean ± SD). The results were statistically analyzed using unpaired t-tests, n = 3. Statistical significance is indicated by * (significant) for the indicated comparisons. Statistics were indicated by *p < 0.05.
environment of periodontal tissue and serve as functional tissue constructs for periodontal regeneration. We anticipate that this work will facilitate the discovery of new strategies for tuning the fate of endogenous resident progenitor cells both for in situ recruitment and for controlled differentiation for periodontal tissue regeneration.
strength causing tissue collapse, and the inability to maintain space.15,16 Therefore, there is a need for bioactive periodontal membranes that are both osteoconductive and biodegradable. Here, we have developed an osteoconductive periodontal membrane with tunable mechanical and degradation characteristics. We have combined multiple physicochemical techniques to expand the properties of this newly developed periodontal membrane. The electrospun membranes were developed using a Federal Drug Administration (FDA)-approved synthetic polymer, poly(ε-caprolactone) (PCL). While this biocompatible polymer has been used for a variety of biomedical applications, to make it suitable for cell adhesion, further surface treatments are required. A number of chemical and physical surface modifications have been reported previously.17 Due to the adhesive properties of polyphenols, polydopamine (PDA), in particular, has attracted significant interest in many fields as a multifunctional thin-film coating.18−20 Polydopamine is a synthetic melanin-like polymer that mimics the composition of the secretion that mussels use to adhere and to attach to surfaces in wet conditions.19,21 Dopamine can self-polymerize at basic pH, which leads to coating of immersed surfaces.20,22 The presence of dopamine-based structures provides favorable adhesive properties, even in wet conditions (vide supra),21 and can accelerate the mineral deposition of hydroxyapatite in the presence of simulated body fluids.23 In addition, this biomimetic polymer can promote cellular adhesion and affect cell fate.21,24 First, to investigate the in vitro functionality of the PDA-coated membrane that we developed for craniofacial bone tissue engineering, we have used several types of dental-derived human mesenchymal stem cells (MSCs) including gingival-derived mesenchymal stem cells (GMSCs), human periodontal ligament stem cells (PDLSCs), and human bone-marrow mesenchymal stem cells (hBMMSCs). Morphological micropatterning approaches have been used as engineering tools to control the localization and fate of mesenchymal stem cells.25,26 Moreover, the membrane that we have developed can be modified with functional proteins including cytokines, growth factors, and extracellular proteins. These adaptations provide precise control of the chemical features of the substrate in accord with physical patterns to regenerate heterogeneous tissues like the periodontium. Here, we have developed membranes with multiscale architectures to enhance periodontal tissue regeneration. Such multiscale designs can mimic the complex extracellular
RESULTS AND DISCUSSION We have utilized electrospinning to fabricate nanofibrous membranes based on PCL (Figure 1a). Solutions of PCL in hexafluoroisopropanol (HFIP) were used for electrospinning under a variety of conditions, including manipulation of the polymer concentration (5−20%), voltage (5−25 kV), and infusion rate (1.0−5.0 mL/h). The optimized conditions were found to be 10% solution at 15 kV with a tip-to-target distance of 8−10 cm and a constant infusion rate of 2.5 mL/h. Changes in fiber morphology were achieved by tuning the electrospinning conditions as proposed previously.27,28 Inspired by mussel adhesive proteins that contain 3,4-dihydroxy-L-phenylalanine (L-DOPA), the developed membranes were coated in a biomimetic fashion using polydopamine. Dopamine can spontaneously self-polymerize at a slightly basic pH (>8).20 The molecular mechanism for the formation of PDA layers and how they interact with supporting layers (here PCL) is poorly understood. We expect that coating here happens by first partially wetting the nanofibrous PCL surface with dopamine monomer with subsequent oxidation of the catechols to quinones, followed by cyclization and structural rearrangement to form the adhesive polydopamine layer, as reported previously for polymeric surfaces.20−24 The oxidative environment also partially oxidizes the PCL surface, which would facilitate formation of the PDA layer. As demonstrated in Figure 1b, this biomimetic approach was utilized to coat the PCL nanofibers and form PDA-coated membranes. Based on scanning electron micrographs (Figure 1c), the presence of PDA does not change the morphology of the fibers. Coated and uncoated PCL nanofibers showed uniform and bead-free nanofibers with average diameters of 270 ± 30 nm and interconnected porous structures with 2.1 ± 0.7 μm diameter pores. Nanofibrous membranes can undergo morphological patterning. Here, we used metal substrates with various mesh sizes to generate membranes with corresponding morphologies (Figure 1d). Template meshes with opening sizes of 20−800 C
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Figure 3. (a) Viability of periodontal ligament stem cells (PDLSCs) cultured on the (left) poly(ε-caprolactone) (PCL) and (right) PCL− polydopamine (PDA) electrospun membranes was checked using (b) a live/dead fluorescence assay at different time points. Scale bars: 100 μm. (c) Analysis of gene expression profiles of osteogenic markers. Real-time polymerase chain reaction (qPCR) analysis of the osteodifferentiation of PDLSCs cultured on PCL and PCL−PDA membranes after 4 weeks in either regular culture or osteogenic media. Expression levels of three osteogenic genes, Col1, RUNX2, and OCN, were evaluated relative to GAPDH, which was used as a reference gene. Morphology of PDLSCs cultured on (d) PCL and (e) PCL−PDA membranes after 4 weeks of culture in osteogenic media evaluated by SEM and presented at different magnifications; from left to right, scale bars represent 200, 20, and 2 μm. Immunofluorescence staining of PDLSCs on membranes with different formulations after culturing in regular media for 2 weeks. (f) PCL−PDA membranes; (g) PCL−PDA membranes pre-treated in simulated body fluid for 48 h before adding stem cells; (h) PCL-PDA membranes pre-treated with rhBMP-2 growth factor (100 ng/mL). β-Actin (green) and DAPI (blue), scale bar: 100 μm. (i) qPCR evaluation of osteogenic gene RUNX2, with reference to the housekeeping gene GAPDH for PDLSCs cultured on PCL−PDA with or without pretreatment in artificial saliva or with rhBMP-2 after 4 weeks of culture in regular or osteogenic media. Values are expressed as mean ± SD. The results were statistically evaluated using unpaired t-tests, n = 3. For all the tests, p < 0.01 was considered statistically significant. Statistics were indicated by *p < 0.05, **p < 0.01, and ***p < 0.001.
μm were used (Figure 1e). In electrospinning, fibers do not follow the patterns of meshes smaller than 150 μm. The coating thickness can be adjusted either by varying the dopamine concentration or by the time of polymerization. We
coated a PCL-based surface at a constant dopamine concentration (2 mg/mL); the time dependence of the coated thickness is shown in Figure 1f. As shown, the coating level reaches a plateau after about 16 h of reaction. X-ray D
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Figure 4. (a) Immunofluorescence staining of human periodontal ligament stem cells (PDLSCs) on patterned membranes shows complete covering of the structures (valleys: upper panels; heals: lower panels) with cells. β-Actin (green) and the dye DAPI (blue), scale bars: 100 μm. (b) Scanning electron micrographs of membranes after 2 weeks in culture in regular media also indicate the ability of cells to remodel the membranes upon their movement and secreting their own extracellular matrix.
photoelectron spectroscopy (XPS) verified the inclusion of the dopamine elements (carbon, nitrogen, and oxygen) in the spectra of the PDA-coated nanofibers (Figure 1g,h). The successful deposition of the PDA coating is indicated by the appearance of the nitrogen (N 1s peak) at 295 eV and reduction in the atomic percentage of oxygen (O 1s peak) after treatment of the PCL surfaces (Figures 1g and S1). Less oxygen is expected on the surfaces after PDA coating since PCL has a 3:1 carbon-to-oxygen ratio, while dopamine has a 4:1 ratio. Both the PCL and PDA-coated PCL (PDA−PCL) membranes degraded in vitro over time (Figure S2). The degradation rates of the coated membranes were slightly faster than the unmodified membrane. The presence of an oxidative agent, such as H2O2, can facilitate degradation of the PDA coating as well as the core PCL, as reflected in increases in degradation rate and decreases in half-life (Figure S2). To suit a wide range of medical and dental applications, regenerative membranes need to provide a range of degradation properties. Simple incorporation of fast degrading polymers (e.g., gelatin or collagen) or using PCL with lower or higher molecular weight into the PCL solution at various concentrations prior to electrospinning can provide different degradation time profiles (Figure S2). Using this manipulation, degradation of these membranes can be tuned to take 2−10 weeks (Figure S2). The mechanical properties of the membranes are affected by coating via PDA as well as the addition of gelatin. Figure 2c shows the tensile properties of membranes. As shown, the tensile strengths and Young’s moduli of the nanofibers were slightly (p < 0.05) enhanced after PDA coating. However, more significant changes (p < 0.01) in these properties can be accomplished when gelatin is included in the formulation (Figure S3). Based on our data, it is possible to increase the modulus over 6-fold (1−6 MPa) to expand the range of biomedical applications of these membranes.
In addition, our data demonstrate that our PDA-modified membrane provides an adherent substrate for cells without affecting their viability (Figure 3a,b). Viability of over 80% was shown for MSCs seeded on a PDA−PCL membrane after 2 weeks in regular culture media. Our data show that engineered nanofibrous substrates can provide optimal rigidity and morphological features that can induce osteogenic differentiation of cultured periodontal ligament stem cells (PDLSCs) in the presence of the PDA coating, consistent with the results of polymerase chain reaction (PCR) analyses (Figure 3c). Changes in the expression of early osteogenic markers collagen type I (Col I) and Runt-related transcription factor 2 (RUNX2), as well as a late osteogenic marker, osteocalcin (OCN), were evaluated. As shown in Figure 3c, the presence of a PDA layer can actively promote osteogenic differentiation of MSCs. Moreover, changing the cell media from regular to osteogenic accelerated the process. After culturing cells in regular or osteogenic media for 4 weeks, the morphology of MSCs seeded on PCL and PDA− PCL was analyzed using scanning electron microscopy. As shown in Figure 3d,e, higher levels of biomineralization were observed in the presence of PDA coatings, consistent with our PCR results. We also examined the effects of preconditioning the membranes with simulated body fluids (and artificial saliva) or a therapeutic protein (e.g., rhBMP-2) on the attachment and differentiation of MSCs (Figure S4). Protein adsorption or hydroxyapatite formation may reduce cellular attachment compared to the intact PDA-coated membrane (Figure 3f−i). As the PDA coating can adsorb ions from the media and initiate mineral deposition, there is not a significant difference in RUNX2 expression levels between the specimens preincubated in simulated body fluids (or artificial saliva) and the intact PDA−PCL. However, the presence of rhBMP-2 can promote osteogenic differentiation of PDLSCs after 4 weeks of culture in regular media compared to other groups (Figure 3i). E
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Figure 5. (a) Periodontal defect model in rat. (b) Rat periodontal defects were created by elevating mucoperiosteal flaps, thereby exposing the alveolar bone crest adjacent to the mesiolingual side of the maxillary first molar. A periodontal window defect was created by removing the alveolar bone covering the root surfaces of a tooth. Membranes with the various formulations were placed into the defect sites. (c) Microcomputed tomography (μCT) analyses of the rat maxilla representing the control (healthy), the defect site without treatment, and the defect size after implantation of the PCL, PCL−PDA, or PCL−PDAPatterned membranes. All specimens were normalized, and μCT images were calibrated for to enable quantitative comparisons. (d) Quantitative analyses of the vertical bone recovery as determined by measuring the distance between the bone crest and cementoenamel junction (CEJ) before and after treatment with the different membrane formulations. The relative volumetric bone recovery was calculated using 3D reconstructed volumes. Data are expressed as average ± SD. The results were statistically analyzed using unpaired t-tests, n = 4. Statistics were indicated by *p < 0.05, ***p < 0.001 between groups and positive controls (no defect); #p < 0.05, ##p < 0.01, ###p < 0.001 between samples and negative controls (no treatment); Δp < 0.05 between indicated samples.
There was no statistical difference in the RUNX2 expression level (p > 0.5) of cells cultured in osteogenic differentiation media. Next, PDA-coated, morphologically patterned PCL membranes were used as cell substrates. As shown in Figure 4a, cell growth followed the patterns; as the PCL core is biodegradable, cells can manipulate their microenvironments as desired. Scanning electron microscopy images (Figure 4b) confirmed that cells actively affect the membrane structure and remodel the membrane even after 2 weeks of culture in regular media. The presence of PDA can accelerate adsorption of calcium and phosphate ions from the media and initiate formation of nanoscale particles of hydroxyapatite (Figure S5a). This intrinsic effect of PDA has been reported previously23 and is promising for bone/dental tissue engineering applications.
After 24 h of incubation of PDA-modified membranes in artificial saliva or simulated body fluids, formation of hydroxyapatite nanoparticles was detected (Figure S5b). To test the in vivo function of the engineered PDA-coated membrane, a periodontal defect was created in rats, and bone regeneration at the defect sites was evaluated 8 weeks postimplantation. The animals were divided into four groups (four rats per group): (i) no treatment (sham), (ii) PCL membrane, (iii) PCL−PDA membrane, or (iv) PCL− PDAPatterned membrane. Subsequently, microcomputed tomography (μCT) was performed to evaluate the amount of regenerated bone (attachment gain) at each defect site. Figure 5c shows μCT images before defect formation and after 8 weeks of treatment with different membrane formulations. The vertical bone gain at each defect site was determined by F
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were obtained from Thermo Fisher Scientific (Waltham, MA, USA), except as indicated otherwise. Stem Cell Isolation and Culture. Mesenchymal stem cell culture: young healthy male individuals undergoing third molar extractions were selected for extraction of gingival and PDL tissues with Institutional Review Board (#BUA6510) approval. The dentalderived stem cells were isolated and then cultured in regular stem cell culture media. Flow cytometry was used to test if MSCs were positive for CD146 and STRO-1 (BD Biosciences, San Jose, CA, USA) surface markers. Cells with passage four (or less) were used in in vitro experiments.30 Electrospinning. Medical grade poly(ε-caprolactone) was obtained from Lactel Absorbable Polymers (Birmingham, AL, USA). Ester-terminated PCL with average molecular weights (Mw) of 80 000 g/mol were used for this study. The PCL polymer (10% w/ w) was dissolved in hexafluoroisopropanol. The 0.5 mL PCL solution per scaffold was electrospun using a laboratory-scale electrospinning device at 20 kV. The constant infusion rate of 2.5 mL/h was used for all experiments. Stainless steel metal meshes with different mesh sizes were used as the substrates for making the membranes with morphological patterning. Polydopamine Coating. Dopamine hydrochloride at a constant concentration of 2 mg/mL was dissolved in basic (pH 8.5) Tris-HCl buffer (10 mM), and membranes were immersed into the solution. The pH-induced self-polymerization of dopamine changes the color of the solution to dark brown. Membranes were incubated in medium at room temperature for different lengths of time in the reaction. The coated membranes were washed with Milli-Q water at least three times and dried with nitrogen gas. The 1× simulated body fluid (SBF) was made by dissolving appropriate quantities of NaCl, NaHCO3, KCl, K2HPO4·3H2O, MgCl2·6H2O, CaCl2, HCl, Na2SO4, and tris(hydroxymethyl)aminomethane (NH2C(CH2OH)3) in Milli-Q water to provide the following ions in appropriate concentration: Ca2+ (2.5 mM), Cl− (148.8 mM), HCO3− (4.2 mM), HPO42− (1 mM), K+ (5 mM), Mg2+ (1.5 mM), Na+ (142 mM), and SO42− (0.5 mM), according to published protocols at 37 °C and the pH was adjusted to 7.4. Membranes were incubated in SBF medium for different lengths of time and then washed using Milli-Q water and dried using nitrogen gas. Characterization. To assess the degradation of membranes in vitro PCL and PDA−PCL membranes (10 mg) were incubated in 0.5 mL 1× phosphate-buffered saline (PBS) at 37 °C for various lengths of time up to 50 days. The degradation was measured by weighing the samples after rinsing in deionized water (twice) and drying overnight. For each group, three to five samples were used per time point. The morphology of nanofibers and cultured cells was characterized using scanning electron microscopy (SEM; Zeiss Supra 40VP) after sputter coating of membranes with an iridium layer using South Bay Technology ion beam sputtering (San Clemente, CA, USA). Elemental surface analysis was performed using XPS (AXIS Ultra DLD, Kratos Analytical Inc., Chestnut Ridge, NY, USA). Spectra were recorded with a 300 ms dwell time and 160 eV for survey spectra and 20 eV for high-resolution spectra of the C 1s, N 1s, and O 1s spectral features using a monochromatic Al Kα X-ray source in ultrahigh vacuum (10−9 Torr). For all XPS scans, 15 kV was applied and 10 mA emission was used. Three scans were recorded for each of the survey spectra, and 10 scans were recorded for each of the high-resolution spectra. The thicknesses of the PDA coatings were evaluated using a scanning probe microscope (Dimension Icon; Bruker, Billerica, MA, USA). The masses of PDA coatings were quantified using a modified microbicinchoninic acid (BCA) assay. The PDA-coated membranes (50 mm2) were treated with 250 μL of micro-BCA working solution, and the absorbance was measured at 562 nm after 1.5 h of incubation at 50 °C.31 To measure the viability of the cultured stem cells on membranes, live−dead assays (calcein AM/ethidium bromide homodimer-1) were performed after 3, 7, and 14 days after culturing cells in regular or
estimating the distance between the alveolar bone crest and cementoenamel junction (CEJ) on the lingual side of the maxillary first molar (Figure 5d). In vivo functionality of the membranes was evaluated 8 weeks postimplantation in a periodontal defect in rats. The results (Figure S2) show that the membranes degrade in 5−10 weeks in vitro. We expect faster degradation in vivo due to the presence of inflammation (acidic conditions) and the dynamics of the mouth environment. Therefore, the location of the membranes was not detectable in μCT images. The volumetric bone gains were calculated using 3D reconstruction of the defect site and the bone volume normalized to the sites without defects. Higher levels of bone gain were observed when PDA-coated PCL membranes (PCL−PDA and PCL−PDAPatterned) were used. These samples showed similar regeneration levels of vertical bone level and volumetric bone formation compared to the controls without any defects (p > 0.1). Implantation of the PDA-based membranes significantly increased the amount of vertical bone gain compared to the sham control group (p < 0.001) as well as PCL membranes without coating (p < 0.05). Furthermore, our data show that the patterning of the engineered membranes did not affect the amount of bone regeneration/gain (p > 0.1). Use of these biomimetic membranes allows immediate closure and regeneration of complex periodontal tissue defects without the negative sequelae associated with nondegradable-based GTRs or mechanically nonrobust collagen films.29 Structured membranes provide mechanical support preventing epithelial invasion and offer ECM-mimicking microstructure to control infiltration and subsequent differentiation of local progenitor cells. The mineralization of the membrane, as induced by the PDA layer, as well as its stiffness, can promote the recovery of lost bone in periodontal tissue. We attribute the in vivo outcome to a combination of both cellular and acellular effects. Altogether, our results demonstrate the effectiveness of the biomimetic membranes in periodontal tissue and bone regeneration. In upcoming work, we are optimizing our engineered periodontal membranes to enable delivery of cytokines/growth factors in the presence or absence of stem cells.
CONCLUSIONS AND PROSPECTS We have described a technological foundation to expand the capabilities of nanofibrous membranes by modulating their properties and characteristics via incorporation of a biomimetic PDA nanoscale coating. We have shown that the fiber formulations can be tuned to provide a broad range of mechanical and degradation profiles. We have utilized these membranes in a periodontal setting for craniofacial tissue engineering. These structures direct the fate of patient-derived dental stem cells toward osteogenic differentiation and show great promise for the treatment of periodontitis. This platform further enables the inclusion of additional biomolecular components to aid healing and to prevent infection, which are now being explored. Together, our results suggest that PDA coating of nanofibrous scaffolds is a promising method for developing multifunctional periodontal membranes. MATERIALS AND METHODS Chemicals and Biologicals. Unless noted otherwise, all chemicals were purchased from Sigma-Aldrich, Inc. (St. Louis, MO, USA). Glassware was acid cleaned overnight and then thoroughly rinsed with Milli-Q water. Cell culture reagents, solutions, and dishes G
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osteogenic culture media. The percentage of live cells was quantified using ImageJ software (NIH, Bethesda, MD, USA). The morphology of cultured stem cells under different conditions was determined using fluorescence staining via FITC-Phalloidin and DAPI (Vector Laboratories, Burlingame, CA, USA). Quantitative real-time PCR assays were used to analyze gene expression. Cultured cells were recovered from membranes after the course of treatment, and RNA was isolated via TRIzol reagent. The RNA was reverse transcribed, and single-stranded cDNA was made using Superscript III cDNA synthesis kits. The relative gene expression was determined using the 2‑ΔΔCt technique, normalizing to the Ct of the reference gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH). The sequences of the primers used in this study are detailed in Table S1. In vivo functionality of membranes was evaluated via a periodontal defect model in rats, as described previously32 and recapped briefly here. Sixteen virgin male and female Sprague−Dawley rats (2-monthold; Harlan Laboratories, Livermore, CA, USA) were used for testing membranes in vivo under approved animal protocols. Mucoperiosteal flaps were elevated to uncover the alveolar bone adjacent to the lingual side of the maxillary first molars. A dental bur was used to remove the alveolar bone capping the tooth’s root surfaces on the lingual side under constant saline irrigation. After creation of a periodontal defect (width: 1.5 mm; length: 3 mm; depth: 2 mm), a PCL, PCL−PDA, or PCL−PDAPatterned membrane was implanted into the defect location. The animals were divided into four groups (four rats per group): (i) no treatment (sham), (ii) PCL membrane, (iii) PCL−PDA membrane, or (iv) PCL−PDAPatterned membrane as shown in Figure 5b. Eight weeks postimplantation, the animals were sacrificed and the amounts of recovered bone at defect sites were evaluated using μCT analysis. All specimens were standardized in terms of measured areas, and μCT images were calibrated to enable quantitative comparisons. The vertical bone loss at each defect site was estimated by determining the space between the alveolar bone crest and CEJ using methods previously described in the literature.32 Three-dimensional reconstructions of the defect sites were captured using Amira software (Thermo Fisher Scientific) and utilized to calculate the volumetric bone fill (attachment gain). The bone volume was normalized to the sites without defects. Statistical Analyses. Data are presented as average values and standard deviations (SDs). The statistical analyses of the experimental data used the Student’s t-test, with a significance level of p = 0.05 for comparisons among samples. The threshold was set to p < 0.01 and p < 0.001 for statistically very significant and extremely significant, respectively
M.M.H.S., P.S.W., and A.M. designed the experiments. M.M.H.S., N.N., T.D.Y., and N.T. conducted the membrane preparation and characterization experiments. M.M.H.S., P.S., and N.T. conducted the cell culture experiments. M.M.H.S., T.A., and A.M. designed and performed the animal studies. All authors performed the data analysis and interpreted the results. M.M.H.S., S.A., T.A., A.K., S.L., P.S.W., and A.M. wrote the manuscript with input from all authors. Notes
The authors declare the following competing financial interest(s): M.M.H.S., T.A., P.S.W., and A.M. have patent applications related to the current study and thus may have related financial interests. The other authors declare no potential conflicts of interest with respect to the authorship and/or publication of this article.
ACKNOWLEDGMENTS This work was supported by grants from the National Institute of Dental, Craniofacial Research (DE023825 to A.M.). N.N. gratefully acknowledges support of a UCLA Dissertation Year Fellowship. We thank Prof. Ben Wu for helpful discussions. REFERENCES (1) Pihlstrom, B. L.; Michalowicz, B. S.; Johnson, N. W. Periodontal Diseases. Lancet 2005, 366, 1809−1820. (2) Polimeni, G.; Xiropaidis, A. V.; Wikesjo, U. M. E. Biology and Principles of Periodontal Wound Healing/Regeneration. Periodontology 2006, 41, 30−47. (3) Chen, F. M.; Zhang, J.; Zhang, M.; An, Y.; Chen, F.; Wu, Z. F. A Review on Endogenous Regenerative Technology in Periodontal Regenerative Medicine. Biomaterials 2010, 31, 7892−7927. (4) Bottino, M. C.; Thomas, V. Membranes for Periodontal Regeneration - A Materials Perspective. Front. Oral Biol. 2015, 17, 90−100. (5) Bottino, M. C.; Thomas, V.; Schmidt, G.; Vohra, Y. K.; Chu, T. M.; Kowolik, M. J.; Janowski, G. M. Recent Advances in the Development of GTR/GBR Membranes for Periodontal Regeneration − A Materials Perspective. Dent. Mater. 2012, 28, 703−721. (6) Zhang, H.; Wang, J.; Ma, H.; Zhou, Y.; Ma, X.; Liu, J.; Huang, J.; Yu, N. Bilayered PLGA/Wool Keratin Composite Membranes Support Periodontal Regeneration in Beagle Dogs. ACS Biomater. Sci. Eng. 2016, 2, 2162−2175. (7) Babo, P. S.; Pires, R. L.; Santos, L.; Franco, A.; Rodrigues, F.; Leonor, I.; Reis, R. L.; Gomes, M. E. Platelet Lysate-Loaded Photocrosslinkable Hyaluronic Acid Hydrogels for Periodontal Endogenous Regenerative Technology. ACS Biomater. Sci. Eng. 2017, 3, 1359−1369. (8) El-Fiqi, A.; Kim, J.-H.; Kim, H.-W. Osteoinductive Fibrous Scaffolds Of Biopolymer/Mesoporous Bioactive Glass Nanocarriers with Excellent Bioactivity and Long-Term Delivery of Osteogenic Drug. ACS Appl. Mater. Interfaces 2015, 7, 1140−1152. (9) Fu, Y.; Liu, S.; Cui, S.-J.; Kou, X.-X.; Wang, X.-D.; Liu, X.-M.; Sun, Y.; Wang, G.-N.; Liu, Y.; Zhou, Y.-H. Surface Chemistry of Nanoscale Mineralized Collagen Regulates Periodontal Ligament Stem Cell Fate. ACS Appl. Mater. Interfaces 2016, 8, 15958−15966. (10) Sam, G.; Pillai, B. R. Evolution of Barrier Membranes in Periodontal Regeneration-″Are the Third Generation Membranes Really Here?″. J. Clin. Diagn. Res. 2014, 8, ZE14−ZE17. (11) Munchow, E. A.; Albuquerque, M. T.; Zero, B.; Kamocki, K.; Piva, E.; Gregory, R. L.; Bottino, M. C. Development and Characterization of Novel ZnO-Loaded Electrospun Membranes for Periodontal Regeneration. Dent. Mater. 2015, 31, 1038−1051. (12) Kaushal, S.; Kumar, A.; Khan, M.; Lal, N. Comparative Study of Nonabsorbable and Absorbable Barrier Membranes in Periodontal
ASSOCIATED CONTENT S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsnano.8b09623. Additional information (PDF)
AUTHOR INFORMATION Corresponding Authors
*E-mail:
[email protected]. *E-mail:
[email protected]. ORCID
Nako Nakatsuka: 0000-0001-8248-5248 Thomas D. Young: 0000-0002-3234-7418 Ali Khademhosseini: 0000-0002-2692-1524 Paul S. Weiss: 0000-0001-5527-6248 Present Address
△ Department of Bioengineering, University of California, San Diego, La Jolla, California 92093-0412, United States.
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DOI: 10.1021/acsnano.8b09623 ACS Nano XXXX, XXX, XXX−XXX
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DOI: 10.1021/acsnano.8b09623 ACS Nano XXXX, XXX, XXX−XXX
