Discussion

The addition of bioceramic nanoparticles in electrospun membranes is a straightforward strategy for the fabrication of functional GBR membranes with improved properties. However, aggregation of nanoparticles and a weak inter- action between the ceramic and polymeric components can occur during the fabrication process, which in turn weakens the mechanical properties of such materials. Here, we developed a composite GBR membrane with significantly enhanced mechanical properties by incorporating high amounts of Si-NPs (i.e.> 30 wt %) without substantial aggregation. A Stöber method was selected to pre- pare the Si-NPs, since previous studies have shown that the physical and chem- ical properties of the nanoparticles could be precisely tuned by this method. Nanometric particles were successfully produced and adequate control over size and shape of the nanoparticles was achieved. The nanoparticles presented a clear spherical shape and homogenous dimensions. Our results showed that Si-NPs were successfully incorporated in the electrospun fibers and aggregation starts to occur for composition S50, although being only substantial for composition S75 (Si-NPs content of 43 wt %). Mechanical properties of the membranes were highly affected by the incorporation of Si-NPs. Tensile strength for composition S25 and S50 and tensile modulus for composition S50 increased significantly with the incorporation of the nanoparticles. In vitro cell culture with MC3T3-E1 osteoblastic cells demonstrated that none of the compositions were cytotoxic, allowing cell adhesion and proliferation. Osteogenic differentiation occurred for cells cultured on all the membranes.

The major goal of this work was to incorporate a high number of nanoparticles in the electrospun fibers, achieving a more homogeneous distribution of the nanoparticles and averting aggregation. A major cause for such aggregation are the particles concentration in solution, particles diameter and attractive interactions between the particles, being these stronger when considering anisotropic particles.[45–47] _{In order to overcome this issue, two strategies were}

adopted. First, Si-NPs were developed using a Stöber sol–gel method[42]_{, which}

enabled adequate control over nanoparticle diameter and size distribution. A second strategy was the use of a mixture of TFE and water, conjugated with a surfactant, as already described by Yang et al.[18] _{TFE is a fluorinated alcohol}

commonly used for the dissolution of polypeptides and polymers, such as PCL. Water was added to increase the dielectric constant of the solution, enabling the production of electrospun fibers without beads or defects. Homogenous electrospun fibers with a smooth surface were produced and, as observed by TEM, Si-NPs are well dispersed in the interior of the fibers up to a Si-NPs weight content of 43 wt%, which is remarkable, especially when compared with similar recent studies.[39, 48–50] _{TFE interacts strongly with the PCL polymeric chains,}

resulting in their swelling and a looser polymeric matrix.[51, 52] _{This effect allows}

the anionic surfactant SDS likely also contributed to the appropriate dispersion of the nanoparticles in the solution. SDS is an amphiphilic molecule, interacting with both hydrophobic and hydrophilic compounds. Karlson et al. explain such effect by a surrounding of the nanoparticles by the hydrophilic group of the surfactant molecules, exposing at the same time their hydrophobic region to the polymeric chains. This way, the surfactant worked as a “bridge” between PCL and Si-NPs.[53, 54] _{The presence of a high number of particles in composition}

S75, combined with their hydrophilic nature led to the hindrance of their dispersion in the PCL solution, resulting in phase separation between silica-rich and PCL-rich regions within the fibers and the formation of aggregates in the interior of the PCL fibers.[40, 55] _{Incorporation of Si-NPs had a significant effect}

on the morphology of the electrospun fibers. When comparing the diameter of the electrospun fibers for the different compositions, a decrease in fiber diameter with increasing Si-NPs content was observed (Figure 1). As apparent from Tables S1 and S2 in the Supplementary Information, the addition of Si- NPs resulted in an increase of the conductivity and viscosity of the solutions. As postulated previously, an increase in conductivity leads to a greater tension in the polymeric jet during electrospinning, resulting in an easier formation of fibers and hence in a decrease in fiber diameter.[3, 5] _{According to several studies,}

an increase in viscosity leads to an increase in fiber diameter.[56, 57] _{However, this}

effect was not observed in our work. A possible explanation is that when Si- NPs were incorporated the increase in conductivity overbalanced the increase in viscosity, leading to the rapid formation of the fiber jet and the production of fibers with a smaller diameter.

The mechanical properties of the membranes were significantly affected by the incorporation of Si-NPs. The tensile modulus of S50 membranes was significantly higher than pure PCL membranes (S0), showing that the incorporation of the Si-NPs was beneficial up to this amount. The actual values of tensile moduli can be explained by the properties of PCL and silica. PCL presents a glass transition temperature (Tg) of approximately -60 °C and polymers subjected to electrospinning have shown to present a lower crystallinity.[58] _{The reduction}

in crystallinity can affect the elastic behavior of the membranes, making them become easily deformed when subjected to tensile forces. S0 membranes are composed solely by PCL, presenting the lowest value of tensile modulus. The incorporation of Si-NPs compensated the low modulus of PCL for compositions S25 and S50. The homogeneous distribution of the nanoparticles inside the electrospun fibers can explain the increase in tensile modulus. Allo et al. claim that the presence of silica could promote cross-linking phenomena among the PCL fibers, due to the bonding between the silanol groups (Si-OH) present in the Si-NPs and the carbonyl groups (C=O) present in PCL.[59] _{In our study, the}

formation of these bonds could not be confirmed by ATR-IR, although specific peaks attributed both to PCL and Si-NPs were detected. When membranes S25 and S50 were subjected to tension, the Si-NPs inside the electrospun fibers likely

3

“hold” the polymeric chains, hindering fiber deformation and increasing the elastic behavior of these membranes.[60]

The addition of Si-NPs led to a significant increase of the tensile strength, achieving more than double the value of S0 for S25 and S50 membranes. Furthermore, compared to several studies focused on the development of PCL- silica based composite membranes, it is noticeable that the values obtained in this study are superior or equal to those reported previously.[34, 40, 61–63] _{This increase in}

tensile strength can be explained by the fact that Si-NPs were distributed along the electrospun fibers for compositions S25 and S50, being mostly separated from each other. Such dispersion of the nanoparticles inside the electrospun fibers resulted in an alignment of the nanoparticles in the interior of the fibers, when these were subjected to tensile forces.

Upon immersion of the membranes in PBS the presence of holes in the fibers from S25, S50, and S75 membranes was observed (Figure 4). Such results demonstrate that dissolution of the Si-NPs occurs through time.[34, 37] _{The release}

of Si-NPs in the PBS potentiates the clinical relevance of such membranes by the increase of silica levels in the medium.[64, 65] _{To obtain better insight into the effect}

of PCL : Si-NP membranes on osteoblastic differentiation, an in vitro culture with osteoblastic MC3T3-E1 cells was performed. An increase in DNA content could be observed for all compositions when comparing earlier times of in vitro culture (3 and 7 days) and later time points (14, 21 and 28 days). Cells were cultured in osteogenic medium and consequently, osteogenic differentiation was expected for all groups. In this study, it was intended to evaluate if the presence of membranes containing silica would affect the osteogenic differentiation rate and levels. ALP levels and activity could be detected for all the compositions. However, a direct effect from the Si-NPs on osteoblastic differentiation could not be determined.

5. Conclusion

The present study aimed to develop electrospun membranes for GBR with improved mechanical and biofunctional properties. Nanometric spherical Si- NPs were produced and successfully incorporated in the PCL fibers, being the nanoparticles well dispersed in their interior up to a PCL : Si-NPs ratio of 100:75 (43 wt%). Tensile modulus and tensile strength improved when Si-NPs were added. Membranes have shown to be cytocompatible although a direct action of the membranes on osteoblastic differentiation could not be observed.

References

[1] S. Ma, Z. Chen, F. Qiao, Y. Sun, X. Yang, X. Deng, L. Cen, Q. Cai, M. Wu, X. Zhang, P. Gao, Guided bone regeneration with tripolyphosphate cross-linked asymmetric chitosan membrane, Journal of Dentistry, 2014. 42(12): p. 1603– 1612.

[2] G. Castillo-Dalí, R. Castillo-Oyagüe, A. Terriza, J. L. Saffar, A. Batista, A. Barranco, J. Cabezas-Talavero, C. D. Lynch, B. Barouk, A. Llorens, A. J. Sloan, R. V. Cayón, J. L. Gutiérrez-Pérez, D. Torres-Lagares, In vivo comparative model of oxygen plasma and nanocomposite particles on PLGA membranes for guid- ed bone regeneration processes to be applied in pre-prosthetic surgery: A pilot study, Journal of Dentistry, 2014. 42(11): p. 1446–1457.

[3] N. Bhardwaj, and S. C. Kundu, Electrospinning: a fascinating fiber fabrica- tion technique, Biotechnology Advances, 2010. 28(3): p. 325–347.

[4] X. Hu, S. Liu, G. Zhou, Y. Huang, Z. Xie, X. Jing, Electrospinning of poly- meric nanofibers for drug delivery applications, Journal of Controlled Release, 2014. 185: p. 12–21.

[5] Q. P. Pham, U. Sharma, A. G. Mikos, Electrospinning of polymeric nano- fibers for tissue engineering applications: a review, Tissue Engineering, 2006.

12(5): p. 1197–1211.

[6] T. J. Sill, and H. A. von Recum, Electrospinning: applications in drug delivery and tissue engineering, Biomaterials, 2006. 29(13): p. 1989–2006.

[7] P. Gentile, V. Chiono, C. Tonda-Turo, A. M. Ferreira, G. Ciardelli, Polymeric membranes for guided bone regeneration, Biotechnology Journal, 2011. 6(10): p. 1187–1197.

[8] M. C. Bottino, V. Thomas, G. Schmidt, Y. K. Vohra, T.-M. G. Chu, M. J. Kow- olik, G. M. Janowski, Recent advances in the development of GTR/GBR mem- branes for periodontal regeneration—a materials perspective, Dental Materials, 2012. 28(7): p. 703–721.

[9] X. Li, X. Wang, T. Zhao, B. Gao, Y. Miao, D. Zhang, Y. Dong, Guided bone regeneration using chitosan-collagen membranes in dog dehiscence-type defect model, Journal of Oral and Maxillofacial Surgery, 2014. 72(2): p. 304.e1–304. e14.

[10] H. Tal, A. Kozlovsky, Z. Artzi, C. E. Nemcovsky, O. Moses, Long-term bio-degradation of cross-linked and non-cross-linked collagen barriers in hu- man guided bone regeneration, Clinical Oral Implants Research, 2008. 19(3): p. 295–302.

[11] W. Ji, F. Yang, H. Seyednejad, Z. Chen, W. E. Hennink, J. M. Anderson, J. J. J. P. van den Beucken, J. A. Jansen, Biocompatibility and degradation char- acteristics of PLGA-based electrospun nanofibrous scaffolds with nanoapatite incorporation, Biomaterials, 2012. 33(28): p. 6604–6614.

[12] M. Simian, C. Dahlin, K. Blair, R. K. Schenk, Effect of different microstruc- tures of e-PTFE membranes on bone regeneration and soft tissue response: a

3

histologic study in canine mandible, Clinical Oral Implants Research, 1999.

10(2): p. 73–84.

[13] N. Donos, L. Kostopoulos, T. Karring, Alveolar ridge augmentation by combining autogenous mandibular bone grafts and non-resorbable membranes, Clinical Oral Implants Research, 2002. 13(2): p. 185–191.

[14] J. Wang, L. Wang, Z. Zhou, H. Lai, P. Xu, L. Liao, J. Wei, Biodegradable poly- mer membranes applied in guided bone/tissue regeneration: a review, Polymers, 2016. 8(4): p. 115.

[15] D. Kaigler, J. A. Cirelli, W. V. Giannobile, Growth factor delivery for oral and periodontal tissue engineering, Expert Opinion on Drug Delivery, 2006.

3(5): p. 647–662.

[16] F.-M. Chen, R. M. Shelton, Y. Jin, I. L. C. Chapple, Localized delivery of growth factors for periodontal tissue regeneration: role, strategies, and perspec- tives, Medicinal Research Reviews, 2009. 29(3): p. 472–513.

[17] H.-Y. Tai, E. Fu, L.-P. Cheng, T.-M. Don, Fabrication of asymmetric mem- branes from polyhydroxybutyrate and biphasic calcium phosphate/chitosan for guided bone regeneration, Journal of Polymer Research, 2014. 21(5): p. 421. [18] F. Yang, S. K. Both, X. Yang, X. F. Walboomers, J. A. Jansen, Development of an electrospun nano-apatite/PCL composite membrane for GTR/GBR applica- tion, Acta Biomaterialia, 2009. 5(9): p. 3295–3304.

[19] E.-J. Lee, S.-H. Jun, H.-E. Kim, Y.-H. Koh, Collagen–silica xerogel nanohy- brid membrane for guided bone regeneration, Journal of Biomedical Materials Research Part A, 2012. 100A(4): p. 841–847.

[20] E. Ye, and X. J. Loh, Polymeric hydrogels and nanoparticles: a merging and emerging field, Australian Journal of Chemistry, 2013. 66(9): p. 997–1007. [21] H. C. Guo, E. Ye, Z. Li, M.-Y. Han, X. J. Loh, Recent progress of atomic layer deposition on polymeric materials, Materials Science and Engineering C, 2017.

70: p. 1182–1191.

[22] C. Dhand, N. Dwivedi, X. J. Loh, A. N. Jie Ying, N. K. Verma, R. W. Beuer- man, R. Lakshminarayanan, S. Ramakrishna, Methods and strategies for the synthesis of diverse nanoparticles and their applications: a comprehensive over- view, RSC Advances, 2015. 5(127): p. 105003–105037.

[23] Z. Li, E. Ye, David, R. Lakshminarayanan, X. J. Loh, Recent advances of us- ing hybrid nanocarriers in remotely controlled therapeutic delivery, Small, 2016.

12(35): p. 4782–4806.

[24] B. M. Teo, D. J. Young, X. J. Loh, Magnetic anisotropic particles: toward remotely actuated applications, Particle & Particle Systems Characterization, 2016. 33(10): p. 709–728.

[25] Q. Q. Dou, C. P. Teng, E. Ye, X. J. Loh, Effective near-infrared photodynamic therapy assisted by upconversion nanoparticles conjugated with photosensitiz- ers, International Journal of Nanomedicine, 2015. 10: p. 419–432.

[26] E. Ellis, K. Zhang, Q. Lin, E. Ye, A. Poma, G. Battaglia, X. J. Loh, T.-C. Lee, Biocompatible pH-responsive nanoparticles with a core-anchored multilayer

shell of triblock copolymers for enhanced cancer therapy, Journal of Materials Chemistry B, 2017. 5(23): p. 4421–4425.

[27] E. Ye, M. D. Regulacio, S.-Y. Zhang, X. J. Loh, M.-Y. Han, Anisotropical- ly branched metal nanostructures, Chemical Society Reviews, 2015. 44(17): p. 6001–6017.

[28] X. J. Loh, T.-C. Lee, Q. Dou, G. R. Deen, Utilising inorganic nanocarriers for gene delivery, Biomaterials Science, 2016. 4(1): p. 70–86.

[29] Q. Dou, X. Fang, S. Jiang, P. L. Chee, T.-C. Lee, X. J. Loh, Multi-function- al fluorescent carbon dots with antibacterial and gene delivery properties, RSC Advances, 2015. 5(58): p. 46817–46822.

[30] C. P. Teng, T. Zhou, E. Ye, S. Liu, L. D. Koh, M. Low, X. J. Loh, K. Y. Win, L. Zhang, M.-Y. Han, Effective targeted photothermal ablation of multidrug re- sistant bacteria and their biofilms with NIR-absorbing gold nanocrosses, Ad- vanced Healthcare Materials, 2016. 5(16): p. 2122–2130.

[31] K. Huang, Q. Dou, X. J. Loh, Nanomaterial mediated optogenetics: oppor- tunities and challenges, RSC Advances, 2016. 6(65): p. 60896–60906.

[32] L. L. Hench, The story of Bioglass®, Journal of Materials Science: Materials in Medicine, 2006. 17(11): p. 967–978.

[33] A. Hoppe, N. S. Güldal, A. R. Boccaccini, A review of the biological re- sponse to ionic dissolution products from bioactive glasses and glass-ceramics, Biomaterials, 2011. 32(11): p. 2757–2774.

[34] Y. Ding, J. A. Roether, A. R. Boccaccini, D. W. Schubert, Fabrication of electrospun poly(3-hydroxybutyrate)/poly(ε-caprolactone)/silica hybrid fiber- mats with and without calcium addition, European Polymer Journal, 2014. 55: p. 222–234.

[35] J. Mota, N. Yu, S. G. Caridade, G. M. Luz, M. E. Gomes, R. L. Reis, J. A. Jansen, X. F. Walboomers, J. F. Mano, Chitosan/bioactive glass nanoparticle composite membranes for periodontal regeneration, Acta Biomaterialia, 2012.

8(11): p. 4173–4180.

[36] J. L. Ryszkowska, M. Auguścik, A. Sheikh, A. R. Boccaccini, Biodegradable polyurethane composite scaffolds containing Bioglass® for bone tissue engineer- ing, Composites Science and Technology, 2010. 70(13): p. 1894–1908.

[37] T. Wakita, A. Obata, G. Poologasundarampillai, J. R. Jones, T. Kasuga, Preparation of electrospun siloxane-poly(lactic acid)-vaterite hybrid fibrous membranes for guided bone regeneration, Composites Science and Technology, 2010. 70(13): p. 1889–1893.

[38] H. C. Schröder, X. H. Wang, M. Wiens, B. Diehl-Seifert, K. Kropf, U. Schloßmacher, W. E. G. Müller, Silicate modulates the cross-talk between os- teoblasts (SaOS-2) and osteoclasts (RAW 264.7 cells): inhibition of osteoclast growth and differentiation, Journal of Cellular Biochemistry, 2012. 113(10): p. 3197–3206.

[39] Y. Ding, Q. Yao, W. Li, D. W. Schubert, A. R. Boccaccini, J. A. Roether, The evaluation of physical properties and in vitro cell behavior of PHB/PCL/sol–gel

3

derived silica hybrid scaffolds and PHB/PCL/fumed silica composite scaffolds, Colloids and Surfaces B: Biointerfaces, 2015. 136: p. 93–98.

[40] N. Ganesh, R. Jayakumar, M. Koyakutty, U. Mony, S. V. Nair, Embedded silica nanoparticles in poly(caprolactone) nanofibrous scaffolds enhanced os- teogenic potential for bone tissue engineering, Tissue Engineering Part A, 2012.

18(17–18): p. 1867–1881.

[41] D. S. Kim, H. B. Park, Y. M. Lee, Y. H. Park, J.-W. Rhim, Preparation and characterization of PVDF/silica hybrid membranes containing sulfonic acid groups, Journal of Applied Polymer Science, 2004. 93(1): p. 209–218.

[42] M. Diba, H. Wang, T. E. Kodger, S. Parsa, S. C. G. Leeuwenburgh, High- ly elastic and self-healing composite colloidal gels, Advanced Materials, 2017.

29(11): 1604672.

[43] M. Kharaziha, M. H. Fathi, H. Edris, Development of novel aligned nano- fibrous composite membranes for guided bone regeneration, Journal of the Me- chanical Behavior of Biomedical Materials, 2013. 24: p. 9–20.

[44] X.-Z. Yan, A. W. G. Nijhuis, J. J. J. P. van den Beucken, K. Both, J. A. Jansen, S. C. G. Leeuwenburgh, F. Yang, Enzymatic control of chitosan gelation for de- livery of periodontal ligament cells, Macromolecular Bioscience, 2014. 14(7): p. 1004–1014.

[45] H. Katepalli, V. T. John, A. Tripathi, A. Bose, Microstructure and rheology of particle stabilized emulsions: effects of particle shape and inter-particle inter- actions, Journal of Colloid and Interface Science, 2017. 485: p. 11–17.

[46] E. Schaer, R. Ravetti, E. Plasari, Study of silica particle aggregation in a batch agitated vessel, Chemical Engineering and Processing: Process Intensifi- cation, 2001. 40(3): p. 277–293.

[47] I. Kim, A. Taghavy, D. DiCarlo, C. Huh, Aggregation of silica nanoparticles and its impact on particle mobility under high-salinity conditions, Journal of Petroleum Science and Engineering, 2015. 133: p. 376–383.

[48] J. S. Fernandes, P. Gentile, M. Martins, N. M. Neves, C. Miller, A. Crawford, R. A. Pires, P. Hatton, R. L. Reis, Reinforcement of poly-L-lactic acid electrospun membranes with strontium borosilicate bioactive glasses for bone tissue engi- neering, Acta Biomaterialia, 2016. 44: p. 168–177.

[49] S.-J. Seo, J.-J. Kim, J.-H. Kim, J.-Y. Lee, U. S. Shin, E.-J. Lee, H.-W. Kim, Enhanced mechanical properties and bone bioactivity of chitosan/silica mem- brane by functionalized-carbon nanotube incorporation, Composites Science and Technology, 2014. 96: p. 31–37.

[50] R. H. Dong, Y. X. Jia, C. C. Qin, L. Zhan, X. Yan, L. Cui, Y. Zhou, X. Y. Jiang, Y. Z. Long, In situ deposition of a personalized nanofibrous dressing via a handy electrospinning device for skin wound care, Nanoscale, 2016. 8(6): p. 3482–3488.

[51] C. J. Luo, E. Stride, M. Edirisinghe, Mapping the influence of solubility and dielectric constant on electrospinning polycaprolactone solutions, Macromole- cules, 2012. 45(11): p. 4669–4680.

[52] M. Dziadek, B. Zagrajczuk, M. Ziabka, K. Dziadek, K. Cholewa-Kowalska, The role of solvent type, size and chemical composition of bioactive glass par- ticles in modulating material properties of poly(ε-caprolactone) based compos- ites, Composites Part A: Applied Science and Manufacturing, 2016. 90: p. 90–99. [53] A. Nesterenko, A. Drelich, H. Lu, D. Clausse, I. Pezron, Influence of a mixed particle/surfactant emulsifier system on water-in-oil emulsion stability, Colloids