Piezoelectric calcium/manganese-doped barium titanate nanofibers with improved osteogenic activity

Highlights

• Bioactive Ca²⁺ and Mn⁴⁺ are individually or co-doped into BaTiO₃ nanofibers.

• The ion-doped BaTiO₃ nanofibers remain comparable piezoelectricity to natural bone.

• The optimized Ca²⁺/Mn⁴⁺ co-doped BaTiO₃ nanofibers achieve significantly improved osteogenic activity.

Abstract

The piezoelectric nature of natural bone tissue makes the use of piezoelectric biomaterials in promoting bone regeneration to be a feasible and attractive strategy. Barium titanate (BaTiO₃) is well-known for its high piezoelectricity and widely studied as bone repairing bioceramic, but its lacking of bioactive ions may compromise its contribution to osteogenesis. Calcium is the richest metallic element in bone mineral, and manganese is an important doping element for hydroxyapatite, therein, Ca²⁺ and Mn⁴⁺ were individually or co-doped into BaTiO₃ nanofibers via sol-gel/electrospinning/calcination technique in this study. Compared to pure BaTiO₃ nanofibers, though the piezoelectric coefficient (d₃₃) of Ca²⁺ and/or Mn⁴⁺-doped BaTiO₃ nanofibers decreased with increase in ion doping amount, it could maintain approx. 0.9–3.7 pC/N and comparable to that of native bone (0.7–2.3 pC/N) at an optimized content. Under the synergistic effect of the released bioactive ions and the material piezoelectricity, the BaTiO₃ nanofibers co-doped with Mn⁴⁺ (2 mol%) and Ca²⁺ (10 mol%) (i.e., the sample 2Mn10Ca-BT) achieved the strongest capacity in enhancing the osteogenic differentiation of bone marrow mesenchymal stromal cells (BMSCs), while showing no cytotoxicity. In summary, bioactive ions-doped BaTiO₃ nanofibers are promising scaffolds for bone tissue engineering, thanks to their acceptable biocompatibility, appropriate piezoelectricity, and improved osteogenic activity.

Introduction

To date, the treatment of bone defects still faces major challenges in achieving satisfactory repairing outcomes [1,2]. Though the tissue engineering strategy has offered a promising solution to meet the challenges, in which, continuous improvements in scaffold materials and designs remain the utmost important task [3,4]. Among the reported numerous biomaterials in relation to this field, piezoelectric polymers and bioceramics have attracted researchers’ interests due to the fact that natural bone inherently displays piezoelectricity with a piezoelectric constant (d₃₃) in the range of 0.7–2.3 pC/N [5]. The implantation of piezoelectric materials can restore the local electrophysiological microenvironment, which will contribute to a fast osteogenesis in situ [[6], [7], [8]]. Compared to other attempts via using growth factors, cytokines or other drugs, the use of piezoelectric biomaterials avoids the uncertainties in drug loading/delivery and bioavailability [9,10].

To enhance bone regeneration, the most popularly studied piezoelectric polymer and bioceramic include polyvinylidene (PVDF) and barium titanate (BaTiO₃), and both of them demonstrate good biocompatibility for cell culture and in vivo applications [8,[11], [12], [13]]. PVDF and its copolymers are non-hydrolyzable for their -C-C- backbone, which necessitates a second surgery to remove the polymeric substrates after defect repairing [14]. As a lead-free piezoelectric bioceramics, BaTiO₃ is promising for implantation since biologic bone is composed of ~70% inorganic component [15,16]. BaTiO₃ particles have been proven to promote new bone formation in vivo, by implantation in femur, skull or tibia defect [6,17,18]. To push forward the practical use of BaTiO₃ in bone repairing, nevertheless, several issues require further improvements. Firstly, it is hard to apply BaTiO₃ particles directly for in vivo implantation in the case of bulky defect. Though the BaTiO₃ powders can be pressed into discs for implantation, the dense structure may be not conducive to cell ingrowth and proliferation [19]. To cope with this issue, a piezoelectric polymer (e.g. PVDF) is usually blended with the BaTiO₃ particles to prepare composite membrane or scaffold [20,21], while the nondegradability of PVDF is liable to cause new concerns. Secondly, the d₃₃ coefficient of dense BaTiO₃ is ~190 pC/N [22], this value mismatches the piezoelectricity of biologic bone. Various factors that influencing the value of d₃₃ include porosity [23], grain size [24], sintering temperature [25] and sintering atmosphere [26]. The denser the compressed BaTiO₃ disc is, the higher its d₃₃ coefficient achieves [27,28]. Thirdly, the inorganic minerals in natural bone are mainly non-stoichiometric hydroxyapaite doped with bioactive microelements [29], while the pure BaTiO₃ is lacking of these bioactive elements (e.g. Ca), which may compromise its contribution to osteogenesis. In view of these points, a properly designed bioactive ion-doped BaTiO₃ porous scaffold is expected to be a possible solution for ameliorating these insufficiencies.

Electrospun nanofibers have been highlighted as porous scaffolds for tissue engineering studies because of their morphology mimicking the collagen fibrous network in native extracellular matrix (ECM) [30,31]. Readily, inorganic nanofibers can be produced via electrospinning and subsequent calcination by adding precursors sol-gel into an electrospinnable polymer solution [32,33]. BaTiO₃ nanofibers are thus able to be produced by preparing sol-gel using Ba-containing compounds and tetrabutyl titanate as precursors, mixing with polyvinylpyrrolidone (PVP) alcohol solution for example, and electrospinning, followed by calcination at high temperature to remove all the organic components [34,35]. The obtained bioceramic nanofibers are self-supporting, and the use of polymer mixing or coating is not indispensable to maintain the structure [33,36]. The d₃₃ coefficient of BaTiO₃ nanofibrous networks should be lower than that of BaTiO₃ block, since porosity will decrease the substrate d₃₃ coefficient as mentioned above, and thus it is possible that the piezoelectrical property of BaTiO₃ nanofibers is liable to match the piezoelectricity of native bone.

For bioceramic materials, ion doping is a common but an effective way to regulate their physicochemical and biological properties [37]. And the precursors sol-gel step involved in the preparation of BaTiO₃ affords the feasibility of ion-doping. For examples, Ahmadi et al. [38] had synthesized barium calcium titanate (Ba_xCa_1-xTiO₃) powders by mixing barium acetate, calcium nitrate tetrahydrate and titanium tetraisopropyl alkoxide to obtain the sol-gel for sintering; Tariverdian et al. [39] obtained barium strontium titanate (Ba_xSr_1-xTiO₃) powders in a similar way. These authors found that both the Ba_xCa_1-xTiO₃ and Ba_xSr_1-xTiO₃ showed promotion effects on the proliferation and differentiation of MG-63 cells, which were ascribed to the material electroactivity and the doped bioactive ions. Theoretically, ion-doping should influence material dielectric constant since the doped ions would replace the Ba²⁺ or Ti⁴⁺, and distort the regularity of the BaTiO₃ lattice in its piezoelectrical tetragonal phase [40,41]. However, it is found that this influence on electrical properties is closely related to the type and content of doping ions, as well as, to single or co-doped of different ions. Commonly, the single ion-doping with bioactive ions such as Ca²⁺, Mg²⁺, and Sr²⁺ obviously reduced the electrical properties of BaTiO₃ [[41], [42], [43], [44]]. However, some reports found that the co-doping of Pr/Mn or Bi/Mn could maintain or even improve the piezoelectric properties of BaTiO₃ ceramics at appropriate contents [45,46].

With these approaches, it is meaningful to carry out studies on the preparation of ion-doped BaTiO₃ nanofibers, which hold strong potentials as novel scaffolds for bone regeneration. Herein, calcium and manganese were selected as the doping elements, which were introduced into the precursors sol-gel individually or together before electrospinning. The primary reason in choosing Ca/Mn for the doping was that they were both the inherent elements in human bone and had been proven to be effective in promoting osteogenesis [47,48]. Another consideration was that Mn might take the place of Ti instead of Ba in BaTiO₃ as proposed by some studies [41,49], while Ca would take the place of Ba. These variations might bring controllability on the piezoelectricity of ion-doped BaTiO₃ nanofibers, thereby, improving their biological performance. To the best of our known, this is the first report on ion-doped BaTiO₃ nanofibers regarding their effectivity in upregulating the osteogenic differentiation of bone marrow mesenchymal stromal cells (BMSCs). Comprehensive characterizations including lattice structure, piezoelectricity, ion release and degradation behaviors, biomineralization, BMSCs proliferation and differentiation evaluations, were conducted to verify the osteogenic bioactivity of Ca/Mn-doped BaTiO₃ nanofibers.