Fabrication and optimization of Nanodiamonds-composited poly(ε-caprolactone) fibrous matrices for potential regeneration of hard tissues

Fabrication and optimization of Nanodiamonds-composited poly(ε-caprolactone) fibrous matrices... Background: Electrospun fibrous matrices are of great importance for tissue engineering and drug delivery device. However, relatively low mechanical strength of the fibrous matrix is one of the major disadvantages. NDs with a positive charge were selected to enhance the mechanical property of a composited fibrous matrix by inducing the intermolecular interaction between NDs and polymer chain. We prepared ND-composited poly (ε-caprolactone) (PCL) fibrous matrices by electrospinning and evaluated their performance in terms of mechanical strength and cell behaviors. Methods: A predetermined amounts of NDs (0.5, 1, 2 and 3 wt%) were added into PCL solution in a mixture of chloroform and 2,2,2-trifluoroethanol (8:2). ND-composited PCL (ND/PCL) fibrous matrices were prepared by electrospinning method. The tensile properties of the ND/PCL fibrous matrices were analyzed by using a universal testing machine. Mouse calvaria-derived preosteoblast (MC3T3-E1) was used for cell proliferation, alkaline phosphatase (ALP) assay, and Alizarin Red S staining. Results: The diameters of the fibrous matrices were adjusted to approximately 1.8 μm by changing process variables. The intermolecular interaction between NDs and PCL polymers resulted in the increased tensile strength and the favorable interfacial adhesion in the ND/PCL fibrous matrices. The ND/PCL fibrous matrix with 1 wt% of ND had the highest tensile strength among the samples and also improved proliferation and differentiation of MC3T3- E1 cells. Conclusions: Compared to the other samples, the ND/PCL fibrous matrix with 1 wt% of ND concentration exhibited superior performances for MC3T3 cells. The ND/PCL fibrous matrix can be potentially used for bone and dental tissue engineering. Keywords: Biodegradable polymer, Composite, Nanodiamond, Electrospinning, Guided tissue engineering Background cell migration and proliferation [4]. Many researchers Electrospun fibrous matrices are very useful in tissue have demonstrated the production of fibrous structures engineering because of their large surface area, high from various organic/inorganic materials and also aspect ratio, porosity, and the presence of very small prepared organic/inorganic composite fibers [5, 6]. Fur- pore structures on the fibers [1–3]. Most importantly, thermore, several groups have investigated composite the topological structure of the electrospun products fibers with a therapeutic agent and functional materials can mimic the extracellular matrix and enhance both using the electrospinning method. Zhang et al. fabricated electrospun biomimetic composited chitosan nanofibers containing hydroxyapatite (HAp) and * Correspondence: choisw@catholic.ac.kr confirmed the bone forming ability as shown by the cell Guk Young Ahn and Tae-Kyung Ryu contributed equally to this work. Department of Biotechnology, The Catholic University of Korea, 43 Jibong-ro, proliferation, mineral deposition and morphology Wonmi-gu, Bucheon-si, Gyeonggi-do 420-743, Republic of Korea © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Ahn et al. Biomaterials Research (2018) 22:16 Page 2 of 8 observation [7]. Li et al. designed and evaluated the Silk increased the alkaline phosphatase (ALP) activity [22]. fibroin fibrous scaffolds containing bone morphogenetic By inspiring our previous results, NDs with a positive protein-2 and HAp nanopowders for bone tissue charge were composited in a fibrous matrix in an effort engineering [8]. Ma et al. reported enzymatic degradable to enhance the mechanical strength and cellular hydrogels based on collagen and alendronate conjugated behaviors on the fibrous matrix. We fabricated HAp nanoparticles. These hydrogels demonstrated ex- ND-composited PCL (ND/PCL) matrices using the elec- cellent biocompatibility and promoted the adhesion and trospinning method for guided tissue engineering and proliferation of MC3T3-E1 cells [9]. Yang et al. were optimized the ND concentrations in terms of the tensile successful in coating electrospun poly(ε-caprolactone) properties of the matrix and the proliferation and differ- (PCL) with a thin layer of calcium phosphate for bone entiation of preosteoblast. We believe that the PCL/ND tissue engineering [10]. Rajzer et al. prepared an electro- matrices have great potential applications for regener- spun bi-layer fibrous scaffold using PCL and gelatin ation of hard tissues. modified with calcium phosphate for bone mineralization [11]. Cao et al. fabricated beta-tricalcium phosphate Methods (β-TCP) composited poly(glycolic acid) three-dimensional Preparation of ND/PCL fibrous matrices scaffolds using solvent casting and particle leaching Fibrous matrices were produced using a horizontal method and evaluated their biocompatibility, osteocon- electrospinning setup [23]. Polycaprolactone (PCL, ductivity, osteogenesis and degradation in vivo [12]. In M = 80,000, Sigma–Aldrich), chloroform (CF, Sigma– general, the materials for the enhancement of osteocon- Aldrich), and 2,2,2-trifluoroethanol (TFE, Sigma-Aldrich) duction have been limited to inorganics such as HAp and were used to fabricate the ND/PCL fibrous matrices. NDs calcium phosphates. with a positive charge (46.0 ± 3.4 mV) were purchased Carbon-based materials, including fullerene, graphene, from Neomond Ltd. (Bucheon, Korea). The NDs were an- carbon nanotubes, graphite, nanohorns and nanodia- alyzed using the Zetasizer (Malvern Instruments Ltd., monds (NDs), have also been investigated for various Worcestershire, UK) to determine their zeta potentials. biomedical applications [13, 14]. Depan et al. investi- For the preparation of the PCL fibrous matrix, PCL solu- gated the biological response of graphene conjugated tion (10 mL, 10 wt%, dissolved in CF:TFE = 8:2) was elec- chitosan scaffolds and reported their higher mechanical trospun onto the aluminum dish using a syringe pump properties, lower degradation rate, and enhanced osteo- (NE-1000, New Era Pump Systems Inc., New York, USA). blast cell growth [15]. Pan et al. prepared the multiwall A high voltage was applied between the spinneret (a 24 G carbon nanotubes/PCL composite scaffolds using solu- needle) and the collector located 15 cm away [24]. The tion evaporation technique and evaluated the prolifera- syringe was horizontally fixed on the syringe pump and tion and differentiation of bone marrow stromal cells the PCL and ND/PCL solutions were electrospun onto an [16]. Recently, NDs, which are carbon-based allotrope aluminum foil collector. The ND concentration of the nanoparticles of truncated octahedral composition, have ND/PCL solution was varied as 0.5, 1, 2, and 3 wt%. In attracted attention as an innovative nanomaterial be- order to ensure that their diameter were similar, the cause of their high biocompatibility, spherical morph- fibers were fabricated under different conditions of ology, high density, surface functionality, and strong appliedvoltage andflowrate[25]. hardness [17]. Many researchers have reported on the excellent non-toxicity and biocompatibility of NDs using Characterization of the ND/PCL fibrous matrix a variety of cells such as epithelial cell, adenocarcinoma Scanning electron microscopy (SEM, S-4800, Hitachi cell, neuroblastoma cell, and so on [18, 19]. Grausova et High-Technologies, Co. Ltd., Tokyo, Japan) was used to al. designed silicon films with NDs and confirmed the characterize the morphologies of the ND/PCL fibrous enhanced adhesion, spreading, viability, growth, and matrices. The diameters of the fibers were calculated maturation of human osteoblast-like MG63 cells on from the SEM images of the samples by analyzing at these films [20]. Zhang et al. fabricated fluorescent least 300 fibers using the ImageJ® software (National In- poly(L-lactic acid)-ND composite thin film and demon- stitutes of Health, Bethesda, USA). The tensile properties strated the enhanced proliferation and differentiation of of the ND/PCL fibrous matrices were analyzed by using osteoblasts on this film as well as an increased mechan- a universal testing machine (UTM, LR 10 K, Lloyd in- ical strength of the scaffold [21]. In view of these inter- struments Ltd., London, UK). The dimensions of the fi- esting applications, a systematic study of the effect of brous matrices were 10 mm × 80 mm × 1 mm (width × NDs used as composite materials for tissue engineering length × thickness). The thickness of the fibrous matrix is crucial. was measured with digital caliper. The extension rate Recently, our group prepared bone-targeted drug was maintained at 5 mm/min at room temperature. The carriers conjugated with NDs and found that NDs itself load cell was 50 N with a gauge length of 50 mm. Ahn et al. Biomaterials Research (2018) 22:16 Page 3 of 8 Cell culture on the fibrous matrices microplate reader. Calcium deposits in the fibrous matri- Four types of the ND/PCL fibrous matrices with ND ces were stained by Alizarin Red S (ARS, Sigma–Al- concentrations of 0.5, 1.0, 2.0, and 3.0 wt% were drich). The cell-seeded fibrous matrices were washed prepared for the cell culture, where the PCL fibrous thrice with PBS, and 1 mL of 40 mM ARS was added to matrices served as a control. Each fibrous matrix was each well and kept at 37 °C for 30 min. Subsequently, cut into square sheets of 10 mm length. Mouse the fibrous matrices were washed with distilled water calvaria-derived preosteoblast (MC3T3-E1) and mouse five times and observed by an inverted microscope fibroblast (NIH/3 T3) were used for cell proliferation on (IX71-F22PH, Olympus, Tokyo, Japan) [28]. the matrices. Prior to cell seeding, the five types of fibrous matrices were sterilized using 70% ethanol for Statistics 24 h, washed with PBS (Welgene) three times, and All experimental data were expressed as means ± dipped in the culture medium for a day. The MC3T3-E1 standard deviation (s.d.). Statistical analysis was evalu- and NIH/3 T3 cells with a density of 1.0 × 10 cells in ated by analysis of variance (ANOVA). The statistical the media were used for cell seeding on to each fibrous significance was set at p < 0.05. matrix and cultured in Dulbecco’s Modified Eagle’s Medium (DMEM, Welgene), which contained 10% fetal Results bovine serum (FBS, Welgene) and 1% antibiotics (peni- To prepare ND/PCL fibrous matrices, a predetermined cillin and streptomycin, Welgene). The fibrous matrices amount of ND powders (0.5, 1, 2, and 3 wt% relative to were maintained in the media for a day until the cells the amount of PCL) was added into the PCL organic so- had adhered to them. The cell-seeded fibrous matrices lution (10 wt%), followed by ultrasonication in an ice were subsequently transferred to 24-well plates, and bath for a homogeneous dispersion. As shown in Fig. 1, maintained in an incubator at 37 °C under a humidified the ND/PCL dispersion and electrospun fibrous matrices atmosphere containing 5% CO . The media were chan- exhibited a darker gray color with the increasing ND ged every 2 days. concentration, which was because of the intrinsic color Cell proliferation was measured using the Cell Count- of NDs. The higher contrast of the ND/PCL fibrous ing Kit-8 (CCK-8, Dojindo, Co. Ltd.) at 1, 3, 5, and 7 days matrices with the higher ND concentrations suggested after cell seeding. The CCK-8 solution (20 μL) was the successful incorporation of NDs in each matrix. added to each well of the 24-well plates containing the Figure 2 shows the SEM images of the resultant PCL cell-seeded fibrous matrices and maintained in an incu- and ND/PCL matrices with different ND concentra- bator for 1.5 h [26]. The sample extracts were trans- tions. The fibers of the PCL and ND/PCL matrices ferred to 96-well plates and their absorbances at 450 nm were randomly deposited. Fiber diameter is one of the were measured by using a microplate reader (Spectra- key factors that affects the properties of fibrous max Plus 384, Molecular Devices, Co. Ltd., Philadelphia, matrices and cell activities [29, 30]. In this work, to USA) [27]. evaluate the effect of ND concentration on the tensile Alkaline phosphatase (ALP) assay was performed at 1, strength and cellular activity, the diameters of PCL 3, and 5 days after seeding on the fibrous matrices. The and ND/PCL fibers were adjusted to approximately fibrous matrices were washed thrice with PBS and then 1.8 μmbychanging the flow rate andappliedvoltage immersed in 1 mL of radio-immunoprecipitation assay in the electrospinning setup, because relatively thick buffer (RIPA buffer, Thermo Scientific). Subsequently, fiber can facilitate cell proliferation [31, 32]. The de- they were stored at − 20 °C for 30 min and centrifuged tailed synthetic conditions and average diameter of for 10 min at 13000 rpm. The supernatant (50 μL) was the fibers are presented in Table 1. transferred to a 96-well plate and 50 μLof p-nitrophenyl Figure 3a shows the representative strain-stress curves phosphate (pNPP, Sigma–Aldrich) solution was added to of the PCL and ND/PCL fibrous matrices. The ND/PCL each well. The plate was kept at 37 °C for 30 min and fibrous matrices with ND concentrations less than after the addition of 50 μL of 3 N NaOH, the absorbance 2 wt% showed more elongation as compared to the PCL at 405 nm was measured using a microplate reader. The fibrous matrix. Note that the ND/PCL fibrous matrix total protein content was measured and the ALP activity with 1 wt% of ND exhibited the most elongated and was calculated by dividing the p-nitrophenol quantita- strongest tensile properties. All the tensile properties, in- tion by protein quantification. The supernatant (2 μL) cluding Young’s modulus (Fig. 3b), tensile strength was transferred to a 24-well plate and 800 μL of distilled (Fig. 3c), and elongation at break (Fig. 3d), increased water and 200 μL of Bio-Rad protein assay solution with the increasing ND concentration up to 1 wt% (Bio-Rad Laboratories) were added to each well. 100 μL and declined as the ND concentration increased of this solution was then transferred to a 96-well plate further. The enhanced tensile strengths were mainly and the absorbance at 595 nm was measured using a attributed to the strong intermolecular interaction Ahn et al. Biomaterials Research (2018) 22:16 Page 4 of 8 Fig. 1 Photographs of the PCL solutions and the corresponding ND/PCL matrices with varying ND concentration (0.5, 1, 2, and 3 wt%). The PCL solution and fibrous matrices were used as a control between NDs and PCL polymer chains [33]. During in proliferation rate when NIH/3 T3 cells were cultured solvent evaporation in electrospinning, the aggrega- on the PCL and ND/PCL fibrous matrices. In contrast, tion of NDs in the ND/PCL fibrous matrices with a the proliferation rate of MC3T3-E1 cells on the ND/ large amount of ND concentration might result in PCL fibrous matrix with 1 wt% of ND was approxi- their reduced tensile properties [34, 35]. The Young’s mately 1.5 times that of the PCL fibrous matrix. The modulus and elongation at break increased approxi- proliferation rates of the MC3T3-E1 cells on the ND/ mately six folds by compositing 1 wt% of NDs, as PCL fibrous matrices with 2 and 3 wt% of ND were compared to the PCL fibrous matrix. lower than that on the PCL fibrous matrix, which is Apart from the tensile properties, in vitro cellular attributed to the strong positive surface charge of those effects of the ND/PCL fibrous matrices were also matrices. evaluated using the NIH/3 T3 and MC3T3-E1 cells. To further verify the positive cellular effect of NDs in MC3T3-E1 (mouse calvaria-derived pre-osteoblast) cell a fibrous matrix, MC3T3-E1 cells were cultured on the lines were chosen as the bone model cells, whereas PCL and ND/PCL fibrous matrices and the ALP activity NIH/3 T3 cells (mouse embryonic fibroblast) were used was measured at 1, 3, and 5 days. As shown in Fig. 5, as normal model cells (control). Fig. 4 shows the cell the ND/PCL fibrous matrix increased the ALP activity proliferation on the PCL and ND/PCL fibrous matrices as compared to the PCL fibrous matrix and the employ- with respect to time. There was no significant difference ment of 1 wt% of ND induced the highest ALP activity. Fig. 2 SEM images of the PCL and ND/PCL fibrous matrices with different ND concentrations. The insets are magnified SEM images and the scale bars are 1 μm Ahn et al. Biomaterials Research (2018) 22:16 Page 5 of 8 Table 1 Fabrication conditions of the PCL and ND/PCL fibrous calcium secreted from the differentiated MC3T3-E1 cells matrices with varying ND concentration (0, 0.5, 1, 2, and 3 wt%). was stained using Alizarin Red S to visualize the calcium The fiber diameter of the PCL and ND/PCL fibrous matrices was deposition on the fibrous matrix (Fig. 6). Red color was determined using ImageJ® software vividly observed on the ND/PCL fibrous matrix with Groups Fiber diameter (μm) Voltage (kV) Flow rate (mL/min) 1 wt% of ND. These results confirmed that the PCL only 1.82 ± 0.12 10 0.008 MC3T3-E1 cells were more differentiated on the ND/ PCL fibrous matrix with 1 wt% of ND and also ND 0.5 wt% 1.93 ± 0.13 8.5 0.02 effectively secreted minerals. ND 1 wt% 1.83 ± 0.16 8 0.03 ND 2 wt% 1.84 ± 0.25 7.5 0.03 Discussion ND 3 wt% 1.80 ± 0.24 6.5 0.03 In this work, NDs with a high positive charge of 46.0 ± 3.4 mV were used for the ND/PCL fibrous By considering Figs. 4 and 5 together, it was concluded matrices. The high positive charge could induce the inter- that the ND/PCL fibrous matrices with 2 and 3 wt% of molecular interactions (e.g., electrostatic interaction) be- ND concentrations exhibited relatively lower prolifera- tween NDs and PCL polymer chains, leading to the tion rates for MC3T3-E1 cells but slightly higher ALP favorable interfacial adhesion in composites. The NDs activities in comparison with the PCL fibrous matrix, were well dispersed in the PCL organic solution with- suggesting that the employment of NDs facilitated the out sedimentation for 5 h due to the nano-scale size ALP activity. In addition to the ALP activity test, and high zeta potential of NDs, which was enough for Fig. 3 a Representative strain–stress curves of the ND/PCL fibrous matrices with different ND concentration (0, 0.5, 1, 2, and 3 wt%). b Young’s modulus, c tensile strength, and d elongation at break of the PCL and ND/PCL fibrous matrices. * Significant difference between the two groups (p < 0.05) Ahn et al. Biomaterials Research (2018) 22:16 Page 6 of 8 Fig. 5 Variation of ALP activities of MC3T3-E1 cultured on the PCL and ND/PCL fibrous matrices (0.5, 1, 2, and 3 wt% ND concentration). The PCL fibrous matrices served as the control. * Significant difference between the two groups (p <0.05) 60 °C) [36]. Therefore, the mechanical properties of PCL-based scaffolds need to be enhanced in order to expand the application area. The ND/PCL fibrous matrix with 1 wt% of ND showed the best tensile properties among the samples. It is hard to directly compare the mechanical properties of other PCL-based fibrous matrix composited with inorganic materials because of the dif- ferences in the fiber diameters and the thickness of the samples. However, some comparisons can be made. The Fig. 4 Proliferation of (a) NIH/3 T3 and (b) MC3T3-E1 cells cultured on the PCL only (control) and ND/PCL fibrous matrices with varying ND concentration (0.5, 1, 2, and 3 wt%). * Significant difference between the two groups (p < 0.05) electrospinning. The fiber diameter of the PCL and ND/PCL fibrous matrices was controlled by the flow rate and applied voltage. Unlike the ND/PCL fibrous matrices, the relatively low flow rate for the fabrication of the PCL fibrous matrix was attributed to the electro- static repulsive force between positive charged NDs and nozzle connected to positive voltage in electrospinning. The PCL-based scaffolds generally had a relatively low mechanical strength as compared to other biodegradable Fig. 6 Representative optical images of the cell/fibrous matrices polymers (e.g., poly(lactic acid), poly(glycolic acid), and cultured on the PCL (control) and ND/PCL fibrous matrices (1 wt% ND polydioxanone), which is because of the intrinsic low concentration) for 1 and 7 days, followed by Alizarin Red S staining glass transition temperature of PCL (approximately − Ahn et al. Biomaterials Research (2018) 22:16 Page 7 of 8 tensile strength and Young’s modulus of the PCL fibrous MC3T3-E1 cells. The ND/PCL fibrous matrix can be po- scaffolds with 6 wt% of nanoclay content were increased tentially used for guided tissue engineering of dental tissue less than two-fold in comparison with the PCL-only and bone. Our next endeavors are focused on the develop- scaffolds [37]. In addition, there was no significant ment of a three-dimensional scaffold composited with difference in the tensile strength between NDs for bone replacement. HAp-composited PCL and PCL-only fibrous matrices, Funding whereas the elongation at break was much increased by This study was supported by the Basic Science Research Program through the employment of HAp [38]. These comparisons the National Research Foundation of Korea (NRF) funded by the Ministry of suggested that the employment of ND as a composited Science, ICT & Future Planning (NRF-2015R1A4A1042350 and 2017R1A2B4008093) and a grant of the Korea Health Technology R&D material can notably increase the mechanical strength Project through the Korea Health Industry Development Institute (KHIDI), and thus enhance the application area of PCL-based fi- funded by the Ministry Health & Welfare, Republic of Korea (HI17C0886). brous matrices. Despite the increased mechanical strength, the application of the ND/PCL fibrous matrices Availability of data and materials For data requests, please contact the authors. would be limited to guided tissue engineering of hard tissue because of their low mechanical strength as Authors’ contributions compared to other poly(lactic acid)-based scaffolds with SWC designed and coordinated the research. GYA primarily conducted a three-dimensional structure. research. TKR participated in the design of the research and helped to draft the manuscript. YRC, JRP and MJL helped to fabricate matrices and cell To evaluate the toxicity of NDs in the fibrous experiments. All authors read and approved the final manuscript. matrix, the NIH/3 T3 cells were cultured on the PCL and ND/PCL fibrous matrices. The NIH/3 T3 cells Ethics approval and consent to participate well proliferated all the fibrous matrices over time Not applicable. and there was no significant difference in the prolifer- Consent for publication ation rate among the PCL and ND/PCL fibrous All authors have consented to the submission of this manuscript for matrices, suggesting the nontoxicity of NDs even at a publication. relatively high concentration upto 3 wt%. The higher proliferation rate on the ND/PCL fibrous matrix with Competing interests The authors declare that they have no competing interests. 1 wt% of ND can be attributed to the fact that the bone cells favored a hard surface for proliferation [39–41]. However, the ND/PCL fibrous matrices with Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in 2 and 3 wt% of ND concentrations exhibited slightly published maps and institutional affiliations. lower proliferation rates compared to the PCL fibrous matrix. 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Fabrication and optimization of Nanodiamonds-composited poly(ε-caprolactone) fibrous matrices for potential regeneration of hard tissues

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Materials Science; Biomaterials
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Abstract

Background: Electrospun fibrous matrices are of great importance for tissue engineering and drug delivery device. However, relatively low mechanical strength of the fibrous matrix is one of the major disadvantages. NDs with a positive charge were selected to enhance the mechanical property of a composited fibrous matrix by inducing the intermolecular interaction between NDs and polymer chain. We prepared ND-composited poly (ε-caprolactone) (PCL) fibrous matrices by electrospinning and evaluated their performance in terms of mechanical strength and cell behaviors. Methods: A predetermined amounts of NDs (0.5, 1, 2 and 3 wt%) were added into PCL solution in a mixture of chloroform and 2,2,2-trifluoroethanol (8:2). ND-composited PCL (ND/PCL) fibrous matrices were prepared by electrospinning method. The tensile properties of the ND/PCL fibrous matrices were analyzed by using a universal testing machine. Mouse calvaria-derived preosteoblast (MC3T3-E1) was used for cell proliferation, alkaline phosphatase (ALP) assay, and Alizarin Red S staining. Results: The diameters of the fibrous matrices were adjusted to approximately 1.8 μm by changing process variables. The intermolecular interaction between NDs and PCL polymers resulted in the increased tensile strength and the favorable interfacial adhesion in the ND/PCL fibrous matrices. The ND/PCL fibrous matrix with 1 wt% of ND had the highest tensile strength among the samples and also improved proliferation and differentiation of MC3T3- E1 cells. Conclusions: Compared to the other samples, the ND/PCL fibrous matrix with 1 wt% of ND concentration exhibited superior performances for MC3T3 cells. The ND/PCL fibrous matrix can be potentially used for bone and dental tissue engineering. Keywords: Biodegradable polymer, Composite, Nanodiamond, Electrospinning, Guided tissue engineering Background cell migration and proliferation [4]. Many researchers Electrospun fibrous matrices are very useful in tissue have demonstrated the production of fibrous structures engineering because of their large surface area, high from various organic/inorganic materials and also aspect ratio, porosity, and the presence of very small prepared organic/inorganic composite fibers [5, 6]. Fur- pore structures on the fibers [1–3]. Most importantly, thermore, several groups have investigated composite the topological structure of the electrospun products fibers with a therapeutic agent and functional materials can mimic the extracellular matrix and enhance both using the electrospinning method. Zhang et al. fabricated electrospun biomimetic composited chitosan nanofibers containing hydroxyapatite (HAp) and * Correspondence: choisw@catholic.ac.kr confirmed the bone forming ability as shown by the cell Guk Young Ahn and Tae-Kyung Ryu contributed equally to this work. Department of Biotechnology, The Catholic University of Korea, 43 Jibong-ro, proliferation, mineral deposition and morphology Wonmi-gu, Bucheon-si, Gyeonggi-do 420-743, Republic of Korea © The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Ahn et al. Biomaterials Research (2018) 22:16 Page 2 of 8 observation [7]. Li et al. designed and evaluated the Silk increased the alkaline phosphatase (ALP) activity [22]. fibroin fibrous scaffolds containing bone morphogenetic By inspiring our previous results, NDs with a positive protein-2 and HAp nanopowders for bone tissue charge were composited in a fibrous matrix in an effort engineering [8]. Ma et al. reported enzymatic degradable to enhance the mechanical strength and cellular hydrogels based on collagen and alendronate conjugated behaviors on the fibrous matrix. We fabricated HAp nanoparticles. These hydrogels demonstrated ex- ND-composited PCL (ND/PCL) matrices using the elec- cellent biocompatibility and promoted the adhesion and trospinning method for guided tissue engineering and proliferation of MC3T3-E1 cells [9]. Yang et al. were optimized the ND concentrations in terms of the tensile successful in coating electrospun poly(ε-caprolactone) properties of the matrix and the proliferation and differ- (PCL) with a thin layer of calcium phosphate for bone entiation of preosteoblast. We believe that the PCL/ND tissue engineering [10]. Rajzer et al. prepared an electro- matrices have great potential applications for regener- spun bi-layer fibrous scaffold using PCL and gelatin ation of hard tissues. modified with calcium phosphate for bone mineralization [11]. Cao et al. fabricated beta-tricalcium phosphate Methods (β-TCP) composited poly(glycolic acid) three-dimensional Preparation of ND/PCL fibrous matrices scaffolds using solvent casting and particle leaching Fibrous matrices were produced using a horizontal method and evaluated their biocompatibility, osteocon- electrospinning setup [23]. Polycaprolactone (PCL, ductivity, osteogenesis and degradation in vivo [12]. In M = 80,000, Sigma–Aldrich), chloroform (CF, Sigma– general, the materials for the enhancement of osteocon- Aldrich), and 2,2,2-trifluoroethanol (TFE, Sigma-Aldrich) duction have been limited to inorganics such as HAp and were used to fabricate the ND/PCL fibrous matrices. NDs calcium phosphates. with a positive charge (46.0 ± 3.4 mV) were purchased Carbon-based materials, including fullerene, graphene, from Neomond Ltd. (Bucheon, Korea). The NDs were an- carbon nanotubes, graphite, nanohorns and nanodia- alyzed using the Zetasizer (Malvern Instruments Ltd., monds (NDs), have also been investigated for various Worcestershire, UK) to determine their zeta potentials. biomedical applications [13, 14]. Depan et al. investi- For the preparation of the PCL fibrous matrix, PCL solu- gated the biological response of graphene conjugated tion (10 mL, 10 wt%, dissolved in CF:TFE = 8:2) was elec- chitosan scaffolds and reported their higher mechanical trospun onto the aluminum dish using a syringe pump properties, lower degradation rate, and enhanced osteo- (NE-1000, New Era Pump Systems Inc., New York, USA). blast cell growth [15]. Pan et al. prepared the multiwall A high voltage was applied between the spinneret (a 24 G carbon nanotubes/PCL composite scaffolds using solu- needle) and the collector located 15 cm away [24]. The tion evaporation technique and evaluated the prolifera- syringe was horizontally fixed on the syringe pump and tion and differentiation of bone marrow stromal cells the PCL and ND/PCL solutions were electrospun onto an [16]. Recently, NDs, which are carbon-based allotrope aluminum foil collector. The ND concentration of the nanoparticles of truncated octahedral composition, have ND/PCL solution was varied as 0.5, 1, 2, and 3 wt%. In attracted attention as an innovative nanomaterial be- order to ensure that their diameter were similar, the cause of their high biocompatibility, spherical morph- fibers were fabricated under different conditions of ology, high density, surface functionality, and strong appliedvoltage andflowrate[25]. hardness [17]. Many researchers have reported on the excellent non-toxicity and biocompatibility of NDs using Characterization of the ND/PCL fibrous matrix a variety of cells such as epithelial cell, adenocarcinoma Scanning electron microscopy (SEM, S-4800, Hitachi cell, neuroblastoma cell, and so on [18, 19]. Grausova et High-Technologies, Co. Ltd., Tokyo, Japan) was used to al. designed silicon films with NDs and confirmed the characterize the morphologies of the ND/PCL fibrous enhanced adhesion, spreading, viability, growth, and matrices. The diameters of the fibers were calculated maturation of human osteoblast-like MG63 cells on from the SEM images of the samples by analyzing at these films [20]. Zhang et al. fabricated fluorescent least 300 fibers using the ImageJ® software (National In- poly(L-lactic acid)-ND composite thin film and demon- stitutes of Health, Bethesda, USA). The tensile properties strated the enhanced proliferation and differentiation of of the ND/PCL fibrous matrices were analyzed by using osteoblasts on this film as well as an increased mechan- a universal testing machine (UTM, LR 10 K, Lloyd in- ical strength of the scaffold [21]. In view of these inter- struments Ltd., London, UK). The dimensions of the fi- esting applications, a systematic study of the effect of brous matrices were 10 mm × 80 mm × 1 mm (width × NDs used as composite materials for tissue engineering length × thickness). The thickness of the fibrous matrix is crucial. was measured with digital caliper. The extension rate Recently, our group prepared bone-targeted drug was maintained at 5 mm/min at room temperature. The carriers conjugated with NDs and found that NDs itself load cell was 50 N with a gauge length of 50 mm. Ahn et al. Biomaterials Research (2018) 22:16 Page 3 of 8 Cell culture on the fibrous matrices microplate reader. Calcium deposits in the fibrous matri- Four types of the ND/PCL fibrous matrices with ND ces were stained by Alizarin Red S (ARS, Sigma–Al- concentrations of 0.5, 1.0, 2.0, and 3.0 wt% were drich). The cell-seeded fibrous matrices were washed prepared for the cell culture, where the PCL fibrous thrice with PBS, and 1 mL of 40 mM ARS was added to matrices served as a control. Each fibrous matrix was each well and kept at 37 °C for 30 min. Subsequently, cut into square sheets of 10 mm length. Mouse the fibrous matrices were washed with distilled water calvaria-derived preosteoblast (MC3T3-E1) and mouse five times and observed by an inverted microscope fibroblast (NIH/3 T3) were used for cell proliferation on (IX71-F22PH, Olympus, Tokyo, Japan) [28]. the matrices. Prior to cell seeding, the five types of fibrous matrices were sterilized using 70% ethanol for Statistics 24 h, washed with PBS (Welgene) three times, and All experimental data were expressed as means ± dipped in the culture medium for a day. The MC3T3-E1 standard deviation (s.d.). Statistical analysis was evalu- and NIH/3 T3 cells with a density of 1.0 × 10 cells in ated by analysis of variance (ANOVA). The statistical the media were used for cell seeding on to each fibrous significance was set at p < 0.05. matrix and cultured in Dulbecco’s Modified Eagle’s Medium (DMEM, Welgene), which contained 10% fetal Results bovine serum (FBS, Welgene) and 1% antibiotics (peni- To prepare ND/PCL fibrous matrices, a predetermined cillin and streptomycin, Welgene). The fibrous matrices amount of ND powders (0.5, 1, 2, and 3 wt% relative to were maintained in the media for a day until the cells the amount of PCL) was added into the PCL organic so- had adhered to them. The cell-seeded fibrous matrices lution (10 wt%), followed by ultrasonication in an ice were subsequently transferred to 24-well plates, and bath for a homogeneous dispersion. As shown in Fig. 1, maintained in an incubator at 37 °C under a humidified the ND/PCL dispersion and electrospun fibrous matrices atmosphere containing 5% CO . The media were chan- exhibited a darker gray color with the increasing ND ged every 2 days. concentration, which was because of the intrinsic color Cell proliferation was measured using the Cell Count- of NDs. The higher contrast of the ND/PCL fibrous ing Kit-8 (CCK-8, Dojindo, Co. Ltd.) at 1, 3, 5, and 7 days matrices with the higher ND concentrations suggested after cell seeding. The CCK-8 solution (20 μL) was the successful incorporation of NDs in each matrix. added to each well of the 24-well plates containing the Figure 2 shows the SEM images of the resultant PCL cell-seeded fibrous matrices and maintained in an incu- and ND/PCL matrices with different ND concentra- bator for 1.5 h [26]. The sample extracts were trans- tions. The fibers of the PCL and ND/PCL matrices ferred to 96-well plates and their absorbances at 450 nm were randomly deposited. Fiber diameter is one of the were measured by using a microplate reader (Spectra- key factors that affects the properties of fibrous max Plus 384, Molecular Devices, Co. Ltd., Philadelphia, matrices and cell activities [29, 30]. In this work, to USA) [27]. evaluate the effect of ND concentration on the tensile Alkaline phosphatase (ALP) assay was performed at 1, strength and cellular activity, the diameters of PCL 3, and 5 days after seeding on the fibrous matrices. The and ND/PCL fibers were adjusted to approximately fibrous matrices were washed thrice with PBS and then 1.8 μmbychanging the flow rate andappliedvoltage immersed in 1 mL of radio-immunoprecipitation assay in the electrospinning setup, because relatively thick buffer (RIPA buffer, Thermo Scientific). Subsequently, fiber can facilitate cell proliferation [31, 32]. The de- they were stored at − 20 °C for 30 min and centrifuged tailed synthetic conditions and average diameter of for 10 min at 13000 rpm. The supernatant (50 μL) was the fibers are presented in Table 1. transferred to a 96-well plate and 50 μLof p-nitrophenyl Figure 3a shows the representative strain-stress curves phosphate (pNPP, Sigma–Aldrich) solution was added to of the PCL and ND/PCL fibrous matrices. The ND/PCL each well. The plate was kept at 37 °C for 30 min and fibrous matrices with ND concentrations less than after the addition of 50 μL of 3 N NaOH, the absorbance 2 wt% showed more elongation as compared to the PCL at 405 nm was measured using a microplate reader. The fibrous matrix. Note that the ND/PCL fibrous matrix total protein content was measured and the ALP activity with 1 wt% of ND exhibited the most elongated and was calculated by dividing the p-nitrophenol quantita- strongest tensile properties. All the tensile properties, in- tion by protein quantification. The supernatant (2 μL) cluding Young’s modulus (Fig. 3b), tensile strength was transferred to a 24-well plate and 800 μL of distilled (Fig. 3c), and elongation at break (Fig. 3d), increased water and 200 μL of Bio-Rad protein assay solution with the increasing ND concentration up to 1 wt% (Bio-Rad Laboratories) were added to each well. 100 μL and declined as the ND concentration increased of this solution was then transferred to a 96-well plate further. The enhanced tensile strengths were mainly and the absorbance at 595 nm was measured using a attributed to the strong intermolecular interaction Ahn et al. Biomaterials Research (2018) 22:16 Page 4 of 8 Fig. 1 Photographs of the PCL solutions and the corresponding ND/PCL matrices with varying ND concentration (0.5, 1, 2, and 3 wt%). The PCL solution and fibrous matrices were used as a control between NDs and PCL polymer chains [33]. During in proliferation rate when NIH/3 T3 cells were cultured solvent evaporation in electrospinning, the aggrega- on the PCL and ND/PCL fibrous matrices. In contrast, tion of NDs in the ND/PCL fibrous matrices with a the proliferation rate of MC3T3-E1 cells on the ND/ large amount of ND concentration might result in PCL fibrous matrix with 1 wt% of ND was approxi- their reduced tensile properties [34, 35]. The Young’s mately 1.5 times that of the PCL fibrous matrix. The modulus and elongation at break increased approxi- proliferation rates of the MC3T3-E1 cells on the ND/ mately six folds by compositing 1 wt% of NDs, as PCL fibrous matrices with 2 and 3 wt% of ND were compared to the PCL fibrous matrix. lower than that on the PCL fibrous matrix, which is Apart from the tensile properties, in vitro cellular attributed to the strong positive surface charge of those effects of the ND/PCL fibrous matrices were also matrices. evaluated using the NIH/3 T3 and MC3T3-E1 cells. To further verify the positive cellular effect of NDs in MC3T3-E1 (mouse calvaria-derived pre-osteoblast) cell a fibrous matrix, MC3T3-E1 cells were cultured on the lines were chosen as the bone model cells, whereas PCL and ND/PCL fibrous matrices and the ALP activity NIH/3 T3 cells (mouse embryonic fibroblast) were used was measured at 1, 3, and 5 days. As shown in Fig. 5, as normal model cells (control). Fig. 4 shows the cell the ND/PCL fibrous matrix increased the ALP activity proliferation on the PCL and ND/PCL fibrous matrices as compared to the PCL fibrous matrix and the employ- with respect to time. There was no significant difference ment of 1 wt% of ND induced the highest ALP activity. Fig. 2 SEM images of the PCL and ND/PCL fibrous matrices with different ND concentrations. The insets are magnified SEM images and the scale bars are 1 μm Ahn et al. Biomaterials Research (2018) 22:16 Page 5 of 8 Table 1 Fabrication conditions of the PCL and ND/PCL fibrous calcium secreted from the differentiated MC3T3-E1 cells matrices with varying ND concentration (0, 0.5, 1, 2, and 3 wt%). was stained using Alizarin Red S to visualize the calcium The fiber diameter of the PCL and ND/PCL fibrous matrices was deposition on the fibrous matrix (Fig. 6). Red color was determined using ImageJ® software vividly observed on the ND/PCL fibrous matrix with Groups Fiber diameter (μm) Voltage (kV) Flow rate (mL/min) 1 wt% of ND. These results confirmed that the PCL only 1.82 ± 0.12 10 0.008 MC3T3-E1 cells were more differentiated on the ND/ PCL fibrous matrix with 1 wt% of ND and also ND 0.5 wt% 1.93 ± 0.13 8.5 0.02 effectively secreted minerals. ND 1 wt% 1.83 ± 0.16 8 0.03 ND 2 wt% 1.84 ± 0.25 7.5 0.03 Discussion ND 3 wt% 1.80 ± 0.24 6.5 0.03 In this work, NDs with a high positive charge of 46.0 ± 3.4 mV were used for the ND/PCL fibrous By considering Figs. 4 and 5 together, it was concluded matrices. The high positive charge could induce the inter- that the ND/PCL fibrous matrices with 2 and 3 wt% of molecular interactions (e.g., electrostatic interaction) be- ND concentrations exhibited relatively lower prolifera- tween NDs and PCL polymer chains, leading to the tion rates for MC3T3-E1 cells but slightly higher ALP favorable interfacial adhesion in composites. The NDs activities in comparison with the PCL fibrous matrix, were well dispersed in the PCL organic solution with- suggesting that the employment of NDs facilitated the out sedimentation for 5 h due to the nano-scale size ALP activity. In addition to the ALP activity test, and high zeta potential of NDs, which was enough for Fig. 3 a Representative strain–stress curves of the ND/PCL fibrous matrices with different ND concentration (0, 0.5, 1, 2, and 3 wt%). b Young’s modulus, c tensile strength, and d elongation at break of the PCL and ND/PCL fibrous matrices. * Significant difference between the two groups (p < 0.05) Ahn et al. Biomaterials Research (2018) 22:16 Page 6 of 8 Fig. 5 Variation of ALP activities of MC3T3-E1 cultured on the PCL and ND/PCL fibrous matrices (0.5, 1, 2, and 3 wt% ND concentration). The PCL fibrous matrices served as the control. * Significant difference between the two groups (p <0.05) 60 °C) [36]. Therefore, the mechanical properties of PCL-based scaffolds need to be enhanced in order to expand the application area. The ND/PCL fibrous matrix with 1 wt% of ND showed the best tensile properties among the samples. It is hard to directly compare the mechanical properties of other PCL-based fibrous matrix composited with inorganic materials because of the dif- ferences in the fiber diameters and the thickness of the samples. However, some comparisons can be made. The Fig. 4 Proliferation of (a) NIH/3 T3 and (b) MC3T3-E1 cells cultured on the PCL only (control) and ND/PCL fibrous matrices with varying ND concentration (0.5, 1, 2, and 3 wt%). * Significant difference between the two groups (p < 0.05) electrospinning. The fiber diameter of the PCL and ND/PCL fibrous matrices was controlled by the flow rate and applied voltage. Unlike the ND/PCL fibrous matrices, the relatively low flow rate for the fabrication of the PCL fibrous matrix was attributed to the electro- static repulsive force between positive charged NDs and nozzle connected to positive voltage in electrospinning. The PCL-based scaffolds generally had a relatively low mechanical strength as compared to other biodegradable Fig. 6 Representative optical images of the cell/fibrous matrices polymers (e.g., poly(lactic acid), poly(glycolic acid), and cultured on the PCL (control) and ND/PCL fibrous matrices (1 wt% ND polydioxanone), which is because of the intrinsic low concentration) for 1 and 7 days, followed by Alizarin Red S staining glass transition temperature of PCL (approximately − Ahn et al. Biomaterials Research (2018) 22:16 Page 7 of 8 tensile strength and Young’s modulus of the PCL fibrous MC3T3-E1 cells. The ND/PCL fibrous matrix can be po- scaffolds with 6 wt% of nanoclay content were increased tentially used for guided tissue engineering of dental tissue less than two-fold in comparison with the PCL-only and bone. Our next endeavors are focused on the develop- scaffolds [37]. In addition, there was no significant ment of a three-dimensional scaffold composited with difference in the tensile strength between NDs for bone replacement. HAp-composited PCL and PCL-only fibrous matrices, Funding whereas the elongation at break was much increased by This study was supported by the Basic Science Research Program through the employment of HAp [38]. These comparisons the National Research Foundation of Korea (NRF) funded by the Ministry of suggested that the employment of ND as a composited Science, ICT & Future Planning (NRF-2015R1A4A1042350 and 2017R1A2B4008093) and a grant of the Korea Health Technology R&D material can notably increase the mechanical strength Project through the Korea Health Industry Development Institute (KHIDI), and thus enhance the application area of PCL-based fi- funded by the Ministry Health & Welfare, Republic of Korea (HI17C0886). brous matrices. Despite the increased mechanical strength, the application of the ND/PCL fibrous matrices Availability of data and materials For data requests, please contact the authors. would be limited to guided tissue engineering of hard tissue because of their low mechanical strength as Authors’ contributions compared to other poly(lactic acid)-based scaffolds with SWC designed and coordinated the research. GYA primarily conducted a three-dimensional structure. research. TKR participated in the design of the research and helped to draft the manuscript. YRC, JRP and MJL helped to fabricate matrices and cell To evaluate the toxicity of NDs in the fibrous experiments. All authors read and approved the final manuscript. matrix, the NIH/3 T3 cells were cultured on the PCL and ND/PCL fibrous matrices. The NIH/3 T3 cells Ethics approval and consent to participate well proliferated all the fibrous matrices over time Not applicable. and there was no significant difference in the prolifer- Consent for publication ation rate among the PCL and ND/PCL fibrous All authors have consented to the submission of this manuscript for matrices, suggesting the nontoxicity of NDs even at a publication. relatively high concentration upto 3 wt%. The higher proliferation rate on the ND/PCL fibrous matrix with Competing interests The authors declare that they have no competing interests. 1 wt% of ND can be attributed to the fact that the bone cells favored a hard surface for proliferation [39–41]. However, the ND/PCL fibrous matrices with Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in 2 and 3 wt% of ND concentrations exhibited slightly published maps and institutional affiliations. lower proliferation rates compared to the PCL fibrous matrix. 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Journal

Biomaterials ResearchSpringer Journals

Published: May 30, 2018

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