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Antibacterial and osteoinductive biomacromolecules composite electrospun fiber
Journal Pre-proofs Antibacterial and osteoinductive biomacromolecules composite electrospun fiber Xuewei Cheng, Qin Wei, Yingao Ma, Rui Shi, Tongtong Chen, Yingbo Wang, Chuang Ma, Yong Lu PII: DOI: Reference:
S0141-8130(19)36134-3 https://doi.org/10.1016/j.ijbiomac.2019.09.156 BIOMAC 13433
To appear in:
International Journal of Biological Macromolecules
Received Date: Revised Date: Accepted Date:
3 August 2019 19 September 2019 23 September 2019
Please cite this article as: X. Cheng, Q. Wei, Y. Ma, R. Shi, T. Chen, Y. Wang, C. Ma, Y. Lu, Antibacterial and osteoinductive biomacromolecules composite electrospun fiber, International Journal of Biological Macromolecules (2019), doi: https://doi.org/10.1016/j.ijbiomac.2019.09.156
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© 2019 Published by Elsevier B.V.
Antibacterial and osteoinductive biomacromolecules composite electrospun fiber Xuewei Cheng a#, Qin Wei b#, Yingao Ma a, Rui Shi a, Tongtong Chen c , Yingbo Wang a*, Chuang Ma d*, Yong Lu c* a
College of Chemical Engineering, Xinjiang Normal University, Urumqi, 830054,
Xinjiang, P. R. China. b
Animal Laboratory Center, Xinjiang Medical University, 393 Xinyi Road, Urumqi
830054, P. R. China. c
Radiology Department, Ruijin Hospital, Shanghai Jiao Tong University School of
Medicine, 197 Ruijin 2nd Road, Shanghai 200025, P. R. China. d
Department of Orthopedics Center, the First Affiliated Hospital of Xinjiang Medical
University, 393 Xinyi Road, Urumqi 830054, P. R. China.
#
These authors contributed equally.
* Corresponding Author: [email protected] [email protected] (Y. L).
(Y.
W);
[email protected]
(C.
M);
Abstract: Bone implant materials have been widely used in bone therapy. However, bone infections caused by bacteria can damage the healing and repairing ability of bone tissue, which remains a major problem to be solved in clinical bone regeneration with implants. In this study, silver nanoparticles (Ag-NPs) were uniformly distributed on the inner of Polylactic acid and Gelatin composite fibers (PLLA and Gel, biological macromolecules) by co-electrospinning for improving anti-infection ability and osseointegration. The optimized experimental conditions for this method were having PLLA/Gel mass ratio of 90:10 and Ag content of 7%. Ag-NPs acted as heterogeneous nucleation sites for promoting the nucleation and growth of calcium phosphorus on the surface of composite fiber. Bone marrow-derived mesenchymal stem cells adhered and proliferated well on the surface of the composite fiber, and the positive fluorescence staining indicated the existence of osteoblasts. Vascular endothelial cells had a good adherence and proliferation on the surface of composite fiber, showing good angiogenic properties. Antibacterial rate of the composite fiber was all over 97% against Monilia albicans, Escherichia coli, Staphylococcus aureus and
Pseudomonas
aeruginosa,
showing
good
antibacterial
property.
A
multi-functional biomacromolecules composite fiber was constructed and shown good bioactivity, osteoinductivity, angiogenic and antibacterial properties. Keywords: Electrospinning, composite fibers, biomacromolecules, osteoinductivity, anti-infection
1 Introduction Bone infection is a difficult problem needs to be overcome in clinical bone treatment [1] as it damages the self-healing ability of bone tissue, leading to bone loss and implantation failure [2]. Therefore, the construction of biomaterials that inhibit bone infection has important practical significance. Bone infections are usually caused by bacteria [3], and the most widely-used treatment methods for bone infections are debridement and systemic antibiotic therapy [4]. However, long-term and excessive antibiotic treatment may increase the risk of bacterial resistance [5], besides burst release issue of antibiotics after implantation [6, 7]. Therefore, reducing the bacterial resistance and improving the anti-infection ability of biomaterials has become an urgent need. Electrospinning can be used to construct fiber structures that are morphologically controllable and able to mimic natural extracellular matrix (ECM) [8]. Inspired by this, an ideal biomimetic environment can be designed for cell adhesion and reproduction to promote new tissue growth. Aurelija et al. [9] found that electrospinning can be used in the development of multilayer scaffolds with different biomacromolecules, in which polylactic acid (PLLA), fibrin, geltatin (Gel) and collagen are appropriate environment materials for cell transplantation and form the inner layer of the multilayer scaffold, while polyimide (PI) film can be used as a barrier to block cell migration from the scaffold. Hiroshi et al. [10] prepared submicron-sized polylactic acid/hydroxyapatite (PLLA/HA) fibers using electrospinning and the fibers showed promoted osteoblast distribution and proliferation in vitro. Shao et al. [11] prepared PLLA/tussah silk fibroin (PLLA/TSF) nanofibers by electrospinning, which possessed excellent mechanical properties and good biocompatibility. Pan et al. [12] incorporated Fe3O4 nanoparticles into PLLA fibers by electrospinning and the composite fibers showed high-level bone healing activity. However, the related studies did not consider infection issues during bone regeneration, which is an urgent problem to be solved in as early as the design and synthesis stage of electrospun bone-like fibers.
PLLA and its composites with inorganic materials have been applied and investigated thoroughly in the field of orthopedics[13, 14] . Johnson et al. [15] mixed nanometer hydroxyapatite (n-HA) with PLLA and polycaprolactone (PCL) and deposited them as nanometer coatings on the surface of magnesium matrix, which effectively reduced the degradation rate in vivo, promoted bone formation and guided bone regeneration, but its antibacterial property was not enough to meet clinical requirements. The long-term antibacterial function of biomaterials has always been highly valued by researchers [16]. Silver nanoparticles (Ag-NPs) have a broad antibacterial spectrum that kills up to hundreds of pathogenic bacteria and viruses [17]. Xu et al. [18]deposited a mixed nano-film composed of tannic acid (TA) and Fe3+ on a Ti substrate, and Ag-NPs was wrapped in the nano-film. They found that Ag-NPs were easily aggregated, reducing its antibacterial activity [19]. Gao et al. [20] added Ag-NPs into PLLA fibers using electrospinning and their studies showed that Ag-NPs formed heterogeneous nucleation sites and improved the bioactivity of the material surface. Yang et al. [21]synthesized PLLA fiber membrane by electrospinning, and uniformly distributed Ag-NPs on the surface of fiber membrane by Michael addition reaction. The study showed that the fiber had good antibacterial property. Therefore, the construction of bioactive and osteoinductive PLLA-based biomacromolecules composite fibers with uniform distribution of Ag-NPs, antibacterial and anti-infective properties is an urgent need. Based on the outstanding broad-spectrum antibacterial property of Ag-NPs, this study has successfully constructed biomacromolecules-based antibacterial and osteoinductive composite fibers with uniformly distributed Ag-NPs on the inner surface of the fibers by electrospinning. Firstly, Ag-NPs can attract bone marrow-derived mesenchymal stem cells (BMSCs) from the surrounding area of the implant to the fracture site [22], and they can enter BMSCs, bind to DNA, activate the expression of genes such as HIF-1α and IL-8 and promote cell proliferation. Besides, Ag-NPs can activate TGF-β/BMP signaling pathway[23, 24], induce osteogenic differentiation of BMSCs and activate endothelial nitric oxide synthase (eNOS) to generate NO free radicals by serine phosphorylation[25]. NO contributes to the
mitogenic effect of several growth factors such as vascular endothelial growth factor (VEGF), enhance the proliferation and migration of endothelial cells and controls angiogenesis[26]. In addition, Ag-NPs have a high reduction potential of 0.7996 V, which can destroy the free energy of bacterial transmembrane proton kinetic storage (proton motive force, PMF)[27] and affect the metabolic reaction of bacteria. The level of reactive oxygen species (ROS) is thus rapidly raised up during the above process, break the steady state of ROS in bacteria and cause them to rupture and die. The synthesis mechanism of antibacterial and osteoinductive PLLA-based multifunctional fiber is shown in Scheme 1.
Scheme 1. Construction mechanism of antibacterial, osteoinductive PLLA-based multifunctional fiber.
2 Materials and methods 2.1 Materials PLLA was purchased from Jinan Daigang Biomaterial Co., Ltd., China. Silver nanoparticles (Ag-NPs) with diameter of less than 100 nm was purchased from
Sigma-Aldrich.
Gelatin
(Gel)
was
from
Beijing
Chemical
Plant,
China.
Trifluoroethanol (TFE) was from Sigma-Aldrich, US. 2.2 Test instruments TL01 Electrospinning Machine was from Shenzhen Tongli Micro-Nano Technology Co., Ltd., China. Scanning electron microscope (SEM, LEO-1430VP) was from Carl Zeiss, Germany. Water contact angle analysis instrument (SDC-200) was from Dongguan Sindin Precision Instrument Co., Ltd., China. Thermogravimetric analysis (TGA) instrument (STA 449F3, heating rate at 3 K·min-1, temperature ranging from 10 °C ~1000 °C) was from NETZSCH, Germany. Atomic Absorption Spectrometer (AAS, Z-2000) was from Hitachi, Japan. Confocal microscope (ECLIPSE Ti) was from Nikon, Japan and microplate reader was from Thermo Fisher Scientific, US. 2.3 Preparation of PLLA/Gel/Ag composite fiber Gel weighing 0.16 g was dissolved in 9 mL of TFE and stirred thoroughly until Gel was completely dissolved. Then 1.44 g of PLLA and various amount of Ag-NPs were added to the solution and stirred thoroughly, configured into electrospinning suspensions with Ag content of 3%, 5%, 7% and 9%. The suspensions were then transferred to 20 mL plastic syringes equipped with medical-grade 6# stainless steel needles, adjusted using a syringe pump, and a high voltage of 18 kV·cm-1 was supplied through a high-voltage DC power source at a constant sampling rate of 3 mL·h-1. Electrospinning fibers were collected on aluminum foil-coated stainless steel rotating mandrel (20 cm × 13 cm) placed 15 cm away from the needle. 2.4 Ion release test Composite fiber was soaked in 40 mL of phosphate buffered saline (PBS, pH=7.4) and incubated at 37 °C for 10 days to test Ag+ release profile. The absorbance of Ag+ in the leachate was measured by AAS (table S1) and Ag+ concentration was calculated from its standard curve, in order to evaluate the physiological stability of the composite fiber.
2.5 Bioactivity test Composite fibers were cut into squares of 20 mm × 20 mm, weighed and placed in 37 °C, 40 mL, 2× simulated body fluid (SBF, table S2) for mineralization (n = 3 for each experimental group). SBF was replaced every 24 hours. Samples were taken out at different mineralization time points (1, 3, 5, 7, 10 days) and soaked in 400 mL of deionized water overnight to remove soluble inorganic ions. Samples were then dried at room temperature for 72 hours, weighed again and spray coated with gold to increase surface conductivity, followed with SEM characterization to observe the surface morphology of the samples. The mineralization quantity of calcium and phosphorus salt on the fiber surface was determined by the difference of fiber mass before and after mineralization. 2.6 In vitro evaluation of primary bone marrow-derived mesenchymal stem cells Bone marrow-derived mesenchymal stem cells (BMSCs) were harvested from the bone marrow of 10 days old SD rats. All animal experiment procedures were carried out in accordance with the Guide for the Care and Use of Laboratory Animals and all animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC) of the First Affiliated Hospital of Xinjiang Medical University. Cell study consisted of 3 experimental groups, including PLLA, PLLA/Gel and PLLA/Gel/Ag (n = 7 for each experimental group). The samples were placed in a culture plate, and BMSCs were seeded on the surface of the samples at a seeding density of 5×104 cell·mL-1 with 3.0 mL of α-MEM medium added to each well, and cultured at 37 °C in a CO2 incubator. After 1, 3, 5, and 7 days of culture, the samples were washed with PBS for three times and followed with fixation and dehydration. Then samples were dried and coated with gold to observe the morphology of the cells. CCK-8 was used to examine cell proliferation at the time point of 1, 3, 5, and 7 days. Briefly, 100 μL of cell suspension was obtained per well (n = 3 per sample), mixed with 10 μL of CCK-8 in a 96-well plate and incubated at 37 °C in a CO2 incubator for 3 hours. The absorbance of each well was obtained by a microplate reader at a wavelength of 450 nm. In addition, FITC staining was performed on the cells after 7
days of culture. Cells were first fixed with paraformaldehyde, then treated with Triton X-100 and blocked with goat serum for 20 min. After that, cells were incubated with FITC at 37 °C for 1.5 hours, and counterstained with DAPI for 5 minutes. Each of the above procedures was followed with three times of PBS wash before the next step. 2.7 In vitro evaluation of primary vascular endothelial cells Vascular endothelial cells (VECs) were harvested from the kidney of newborn (3 days old) SD rats. All animal experiment procedures were carried out in accordance with the Guide for the Care and Use of Laboratory Animals and all animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC) of the First Affiliated Hospital of Xinjiang Medical University. Cell study consisted of 3 experimental groups, including PLLA, PLLA/Gel and PLLA/Gel/Ag (n = 7 for each experimental group). The samples were placed in a culture plate, and VECs were seeded on the surface of the samples at a seeding density of 1.5×104 cell·mL-1 with 3.0 mL of α-MEM medium added to each well, and cultured at 37 °C in a CO2 incubator. After 1, 3, 5, and 7 days of culture, samples were washed with PBS for three times, fixed and dehydrated, dried and coated with gold to observe the morphology of the cells. CCK-8 was used to examine cell proliferation at each time point with the similar procedure as samples cultured with BMSCs. 2.8 Antibacterial property test The strains used for antibacterial test were Monilia albicans (M. albicans), Escherichia coli (E. coli), Staphylococcus aureus (S. aureus) and Pseudomonas aeruginosa (P. aeruginosa). All strains were tested in three experimental groups, including PLLA/HA, PLLA/Gel and PLLA/Gel/Ag (n = 3 for each experimental group). For the qualitative analysis, stains of M. albicans, E. coli, S. aureus and P. aeruginosa were inoculated on MH medium separately for activation culture. Samples of each strain were acquired and suspended in separate physiological saline to prepare 10 mL bacterial suspensions with a concentration of 0.5 McFarland unit (0.5 MCF, 1.5×108 cell·mL-1). Then 50 mL of each suspension was obtained, seeded
on a plate and cultured at 37 °C in an incubator for 24 hours. For the quantitative analysis, the strains were inoculated on MH medium for three times to obtain purified colonies. The samples to be tested were sterilized with ultraviolet light for 1 hour before transferred to a glass slide in a Petri dish. In order to prevent the evaporation of the bacterial suspension, sterile water was added to cover the bottom of the Petri dish. Single bacteria colonies were scraped off with inoculating loop and prepared into suspension of 0.5 MCF using physiological saline. 50 μL of the as prepared bacterial suspension was dropped onto the surface of the sample, and cultured in a 37 °C incubator for 24 hours. Afterwards, 20 μL of the remaining bacterial suspension on the surface of the sample was obtained and seeded on a plate, and the number of colonies was counted after another 24 hours of culture. The antibacterial property of the material was represented by antibacterial rate. Three parallel experiments were performed to calculate the average antibacterial rate. The calculation method is shown in equation (1), ݁ݐܽݎ݈ܽ݅ݎ݁ݐܾܿܽ݅ݐ݊ܣൌ
ீିாீ ீ
ൈ ͳͲͲΨ
˄1˅
where CCCG is the colony count of the control group and CCEG is the colony count of the experimental group. 2.9 Statistical analysis A One Sample t Test (assuming unequal variance) was used for statistical analysis and all data are presented as mean ± standard deviation. The difference between two sets of data is considered statistically significant when p < 0.05.
3 Results and discussions 3.1 Preparation of composite fibers Fig. 1 shows the morphology, average diameter, contact angle, surface energy, weight loss and Ag+ release profile of the biomacromolecules composite fibers. It can be seen from Fig. 1a that when the mass ratio of PLLA/Gel was 100/0, the fiber
morphology was inconsistent and fibers were relatively thick. While with the addition of Gel and the mass ratio of PLLA/Gel changed to 90/10 and 80/20, the fiber morphology became relatively homogeneous. However, beads appeared in the fibers when the mass ratio of PLLA/Gel changed to 70/30 (Fig. S1). This was because the addition of Gel has increased the viscosity of the electrospinning solution, causing a continuous increase of surface tension that the electric field force was insufficient to overcome, resulting in uneven fiber diameters during electrospinning process. The average diameters of the PLLA/Gel composite fibers prepared at mass ratio of 100/0, 90/10, 80/20 and 70/30 were 909 nm, 373 nm, 592 nm and 246 nm, respectively (Fig. 1b). Studies have shown that the addition of Gel could increase surface contact angle and thus improving the hydrophilicity of the composite materials due to the rich content of hydroxyl and hydrophilic groups in Gel[28]. According to the water contact angle data of PLLA/Gel (Fig. 1c), the surface contact angle of the composite fiber was 139.2°, 144.1°, 135.9°, and 138.2° with the addition of Gel. According to literature report[29], the hydrophobicity of electrospun fibers increase due to special fiber structure, and the size of the fiber also affects the hydrophobicity of the material. When Gel was added to pure PLLA fiber, the average fiber diameter decreased from 909 nm to 373 nm, and the water contact angle of 90/10 composite fiber was slightly larger than that of pure PLLA fiber. With the increase of Gel addition, the surface water contact angle of 80/20 composite fiber was slightly small than 90/10 group, indicating that the addition of Gel had an effect on the water contact angle, but the effect was not significant[30]. However, when PLLA/Gel mass ratio increased to 70/30, the water contact angle of the composite fiber increased slightly when compared to 80/20 composite fiber, which was due to the smallest diameter of the 70/30 composite fiber. The above results were consistent with Wenzel's theory that the combination of low surface energy and high surface roughness produces a hydrophobic surface (Fig. 1d)[31].
Fig. 1 The (a) morphology, (b) average diameter, (c) water contact angle and (d) surface energy of PLLA/Gel composite fiber.
As Ag is considered to be an ideal inorganic antibacterial agent and able to promote the growth and proliferation of BMSCs [32], Ag-NPs were added to PLLA/Gel (90/10) fibers to construct an antibacterial composite fiber. The surface of the composite fiber with different content of Ag-NPs was smooth, and Ag-NPs were evenly distributed in the fiber. It can be seen from Fig. 2a that no droplets were found on the surface of 3%, 5% and 7% Ag-NP-containing fibers and the diameter of the fibers decreased but the distribution was relatively uniform with the increase of Ag-NPs content. However, the fibers became disordered and easily broken when Ag-NPs content increased to 9% (Fig. S2). This was because the high concentration of Ag-NPs resulted in a low viscosity of the electrospinning liquid, and the electrical conductivity of Ag-NPs made fibers easily stretched discontinuously to break during the electrospinning process. The average diameter of PLLA/Gel/Ag composite fiber (Fig. 2b) was 389 nm, 326 nm, 282 nm and 328 nm for Ag-NPs content of 3%, 5%, 7% and 9%, respectively. The average diameter of the composite fiber decreased along with the increase of Ag-NPs content from 3% to 7%. This was caused by the dropped viscosity of the electrospinning liquid with continues addition of Ag-NPs, the solvent volatilization was accelerated, and the surface tension of the fiber was reduced to easily get stretched under the same electric field force. When the content of Ag-NPs
increased to 9%, a discontinuous and uneven distribution of fibers lead to an increase in the average diameter of the biomacromolecules composite fibers. The hydrophilicity test of the fibers (Fig. 2c) showed that the water contact angles were 143.3°, 144.3°, 138.3° and 138.5° with increasing Ag-NPs content of 3%, 5%, 7% and 9%, respectively. It can be seen from the test result that the water contact angle of composite fibers increased slightly when Ag-NPs content increased from 3% to 5% and then decreased when Ag-NPs content increased to 7%. When Ag-NPs content continued to increase to 9%, the fibers were discontinuous and unevenly distributed, with slightly increased average diameter and water contact angle. The surface energy test of the composite fibers showed that the surface energy of the fiber with 7% Ag-NPs content was the highest (Fig. 2d). From the above characterizations including morphology, average diameter and hydrophilicity of the composite fibers, it was known that the fiber with 7% Ag-NPs addition was the optimum group in this study. The tested Ag-NPs content was 4.4% compared to the designated 7% in the composite fiber (Fig. 3a), and the Ag+ release was stable (Fig. 3b).
Fig. 2 The (a) morphology, (b) average diameter, (c) water contact angle, (d) surface energy of PLLA/Gel /Ag composite fiber.
Fig. 3 The (a) TGA result and (b) Ag release profile of PLLA/Gel/Ag composite fiber.
3.2 Bioactivity of the composite fibers Bioactivity is an important criterion of materials for the integration and repair of human bone tissue. Fig. 4 is a graph showing the bioactivity of the biomacromolecules composite fiber surface after soaked in SBF for mineralization. It can be seen from Fig. 4a that when the PLLA/Gel mass ratio was 100/0, the surface of the fiber was partially coated with nanoparticles after 10 days of mineralization, and the nanoparticles had a tendency to join into pieces. When the mass ratio of PLLA/Gel was 90/10, the surface of the biomacromolecules composite fiber was all covered with nanoparticles. However, the surface of the composite fiber with PLLA/Gel mass ratio of 80/20 had less calcium and phosphorus salt deposition and almost no deposition on the surface of PLLA/Gel fibers with mass ratio of 70/30. With the increase of soaking time, the deposition amount of the nanoparticles on PLLA/Gel fiber surface has increased (Fig. 4c). On the first 5 days, there was not much increase in the deposition of calcium and phosphorus salt on the fiber surface, but difference showed up after 5 days of mineralization, and the deposition of 90/10 composite fiber was the highest. Studies have shown that, the more the Gel content, the stronger the hydrophilicity of the biomacromolecules composite fiber, which was not advantageous for the deposition of calcium and phosphorus salt on the fiber surface. Therefore, the composite fiber with PLLA/Gel mass ratio of 90/10 was the best in this study. It can be seen in Fig. 4b that after 10 days of mineralization, there was no calcium and phosphorus salt deposition on the fiber surface with 3% Ag-NPs addition,
and only small amount of deposition on the fiber surface with 5% Ag-NPs addition. The calcium and phosphorus salt deposition was the highest on the fiber surface with 7% Ag-NPs addition and the fibers were wrapped all over by salt to form a uniform spread. However, the calcium and phosphorus salt only deposited on the surface of the fiber with 9% Ag-NPs addition but not covered the whole surface. Overall, the salt deposition quantity increased with the expanded soaking time, and decreased with the enlarged fiber diameter, among which the deposition of calcium and phosphorus salt reached the maximum on the surface of composite fiber with 7% Ag-NPs addition on day 10 (Fig. 4d). The mineralization mechanism was analyzed accordingly. First of all, the fiber surface had interactions with SBF at the initial stage of soaking and PLLA hydrolyzed some functional groups to produce carboxyl groups. Secondly, the carboxyl groups on the surface of the fiber attracted Ca2+ to form calcium and phosphorus salt granules, which acted as nucleation sites to form a coarse calcium and phosphorus salt layer as the soaking time increased, providing a larger nucleation space for the further deposition of calcium and phosphorus salt. In the meanwhile, Ag-NPs acted as heterogeneous nucleation sites and aided the increase of deposition quantity of calcium and phosphorus salt on the surface of composite fibers. Therefore, the deposition of calcium and phosphorus salt on the surface of composite fibers was the result of the synergistic effect of heterogeneous nucleation of Ag-NPs and composite fiber diameter[19]. The above analysis
demonstrated that the
PLLA/Gel/Ag composite fiber with 7% Ag-NPs addition had the best bioactivity. In summary, the optimal experimental conditions were PLLA/Gel mass ratio of 90/10 and Ag-NPs content of 7%. The following experiments including in vitro cell culture and antibacterial test of the composite fibers were conducted under these conditions.
Fig. 4 Mineralization of composite fibers. (a) The bioactivity of PLLA/Gel composite fiber (SEM). (b) The bioactivity of PLLA/Gel/Ag composite fiber (SEM). (c) Mass increase of PLLA/Gel composite fiber incubated in SBF for 10 days. (d) Mass increase of PLLA/Gel/Ag composite fiber incubated in SBF for 10 days.
3.3 In vitro cell compatibility of composite fibers Fig. 5 shows the morphology, cell viability and fluorescence staining of BMSCs cultured on the surface of composite fibers for 7 days. The CCK-8 result (Fig. 5d) showed that cells adhered well on the surface of different materials and cell densities were close after inoculated on the surface of the fibers for one day. After 3 days, the cells were adapted to the surrounding environment of the material and began to grow. When the cells were cultured for 5 days, they began to proliferate and BMSCs had the best cell activity on the PLLA/Gel/Ag fiber surface when cultured for 7 days. Besides, BMSCs showed a spread morphology on the surface of PLLA, PLLA/Gel and PLLA/Gel/Ag composite fibers on day 7 (Fig. 5a, b) and BMSCs density was the highest on the surface of PLLA/Gel/Ag composite fibers (Fig. 5c). The fluorescence staining of BMSCs on PLLA/Gel/Ag composite fibers was positive (Fig. 5e), which indicated the differentiation of BMSCs into
osteoblasts. Studies have shown that Ag-NPs could promote the proliferation and differentiation of BMSCs, therefore, PLLA/Gel/Ag composite fibers could also promote the proliferation of BMSCs and induce its osteogenesis, showing good osteoinductivity. The mechanism of inducing osteogenesis was that after the cells adhered to the surface of the composite fibers, Ag-NPs attracted the MSCs around the implant to the fracture site and entered MSCs[22], bind to DNA and activated gene expression of HIF-1α and IL-8. This process has promoted cell proliferation, and Ag-NPs could induce and activate TGF-β/BMP signaling pathway, inducing osteogenic differentiation of MSCs[23, 24].
Fig. 5 BMSCs morphology and density cultured on the surface of composite fibers. SEM micrographs showing the morphology of BMSCs cultured on (a) PLLA, (b) PLLA/Gel and (c) PLLA/Gel/Ag surface for 7 days. (d) CCK-8 data plot showing the density of BMSCs cultured on each group of composite fibers for 1, 3, 5 and 7 days. (e) Fluorescence staining of BMSCs cultured on PLLA/Gel/Ag surface on day 7.
The human bone contains a large number of microvessels for the transport of nutrients, so the cytocompatibility of composite fibers with VECs were tested in this study. Fig. 6a is an SEM micrograph of VECs cultured on the surface of composite fibers for 1, 3, 5, and 7 days. The micrograph showed that VECs began to adhere to the surface of the composite fiber, and their pseudopods were extended after 1 and 3 days of culture. Then after 5 and 7 days, VECs began to proliferate and spread on the surface of the composite fiber. Cell activity was tested using CCK-8 and the
result showed (Fig. 6b) that as the culture time continued to increase, the number and activity of VECs has increased. Cell activity was the highest on PLLA/Gel/Ag surface at different culture time points. This was because that a certain concentration of Ag-NPs could stimulate the production of NO by eNOS in cells, and NO could contribute to the mitogenic effect of several growth factors such as VEGF, beneficial for
vascularization[25,
26].
PLLA/Gel/Ag
composite
fiber
has
good
cytocompatibility with VECs and it can promote the growth as well as proliferation of VECs, inducing angiogenesis.
Fig. 6 VECs morphology and density cultured on the surface of composite fibers.(a) SEM micrographs of VECs cultured on composite fibers for 1, 3, 5 and 7 days.(b) CCK-8 data plot of VECs cultured on composite fibers for 1, 3, 5 and 7 days.
3.4 Antibacterial property of composite fibers Gram-negative and Gram-positive bacteria that are commonly seen in clinical treatment, including M. albicans, E. coli, S. aureus, and P. aeruginosa were used in this antibacterial study. It can be seen from Fig. 7a that after 24 hours of incubation, E.
coli and P. aeruginosa on the surface of PLLA fiber were undergoing strong metabolism and reproduction activity, while S. aureus and P. aeruginosa were more active on reproduction on the surface of PLLA/Gel. There were almost no colonies in the medium of the PLLA/Gel/Ag group and that was because the addition of Ag-NPs has enhanced the antibacterial property of the composite fiber. Quantitative analysis (Fig. 7b) showed that the antibacterial rates of PLLA against M. albicans and S. aureus were 80.7% and 74.2%, respectively, and there was no antibacterial activity against E. coli and P. aeruginosa. PLLA/Gel had an antibacterial rate of 90.6% against E. coli but no antibacterial ability against all the other three bacteria strains. With the addition of Ag-NPs, PLLA/Gel/Ag had antibacterial rates over 97% for all four strains. The results showed that PLLA/Gel/Ag composite fiber has good antibacterial ability against E. coli, S. aureus, P. aeruginosa and M. albicans. The main mechanism of the antibacterial activity of Ag-NPs is the metal ion effect and catalysis action. First of all, the cell membrane of Gram-positive and Gram-negative bacteria is negatively charged[33], and it is easy to attract positively charged Ag+ due to the action of static electricity, inactivate cell enzymes and destroy the integrity of cell membrane. In addition, Ag+ interacts with thiol groups in proteins to promote the release of oxygen-active substances, which can cause damage to proteins and DNA, leading to cell death[34]. Secondly, the chemical structure of Ag-NPs provides a strong catalytic ability and a high reduction potential, resulting in the production of ROS in cells, causing bacteria death by strong oxidizing properties[32]. Besides, bacteria use PMF, established by a proton pump, to produce adenosine triphosphate (ATP) through ATP synthase. However, Ag-NPs can destroy PMF, oppress and even disrupt the metabolic reactions of bacterial cells, rapidly increase the level of ROS in bacteria and thus cause death[27]. In addition, Ag-NPs can migrate out of dead bacteria and repeat the above cycle, which is why the antibacterial activity of Ag-NPs is significantly sustainable. Although the membrane structure of Gram-positive and Gram-negative bacteria is different, most bacteria are negatively charged. Therefore, Ag+ can cause bacteria death by electrostatic attraction. Literature has reported that the antibacterial activity of Ag-NPs may be related to the
characteristics of the bacterial species[35]. The outer cell of Gram-negative bacteria has a layer of lipopolysaccharide, followed by a thin layer of peptidoglycan, while the cell wall of Gram-positive bacteria is mainly composed of a thick layer of peptidoglycan. Studies have shown that Ag-NPs have better antibacterial properties against Gram-negative bacteria.
Fig. 7 (a) Qualitative analysis of the antibacterial property of composite fibers. (b) quantitative analysis of the antibacterial property of composite fibers.
4 Conclusions This study has successfully prepared PLLA/Gel/Ag composite fibers with good bioactivity,
osteoinductivity,
angiogenicity
and
anti-infective
property
by
electrospinning on the basis of rich hydrophilic groups in Gel and antibacterial property of Ag-NPs. The results showed that a uniform morphology of the biomacromolecules composite fiber was obtained when the mass ratio of PLLA to Gel
was 90:10 and the content of Ag-NPs was 7%. The rich hydrophilic groups -OH and -NH2 in Gel has improved the adsorption capacity of the composite fiber, and Ag-NPs acted as heterogeneous nucleation sites, promoted the nucleation and growth of calcium and phosphorus salt on the surface of the fiber, and improved the bioactivity of the fiber together with Gel, showing synergistic function. BMSCs adhered and proliferated well on the surface of PLLA/Gel/Ag composite fibers, and the fluorescence staining of cells was positive, indicating osteogenesis of BMSCs. VECs also adhered and proliferated well on the surface of the composite fiber, indicating good angiogenic property. Besides, the antibacterial rate of PLLA/Gel/Ag composite fiber was as high as over 97% against all four strains including M. albicans, E. coli, S. aureus and P. aeruginosa. In this study, electrospinning technology was used to successfully construct multifunctional composite fibers with good bioactivity, osteoinductivity, angiogenic and antibacterial properties. Therefore, PLLA/Gel/Ag composite fibers are potentially viable bone tissue repair materials that can inhibit bone infection and promote osseointegration.
Acknowledgments This work was supported by the National Natural Science Foundation of China (51662038, 81560350 and 81760397), Outstanding Youth Natural Science Foundation Project of Xinjiang (2017Q002), Research and Innovation Project of Postgraduates in Autonomous Region at 2019. Xinjiang Normal University 2018 College Students Innovative
and
Entrepreneurship
Training
Project
(201810762007
and
201810762013), Supported by the “13th Five-Year” Plan for Key Discipline Chemistry, Xinjiang Normal University.
Competing interests We declare that we have no conflicts of interest.
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Figure captions Scheme 1. Construction mechanism of antibacterial, osteoinductive PLLA-based multifunctional fiber.
Fig. 1 The (a) morphology, (b) average diameter, (c) water contact angle and (d) surface energy of PLLA/Gel composite fiber.
Fig. 2 The (a) morphology, (b) average diameter, (c) water contact angle, (d) surface energy of PLLA/Gel /Ag composite fiber.
Fig. 3 The (a) TGA result and (b) Ag release profile of PLLA/Gel/Ag composite fiber.
Fig. 4 Mineralization of composite fibers. (a) The bioactivity of PLLA/Gel composite fiber (SEM). (b) The bioactivity of PLLA/Gel/Ag composite fiber (SEM). (c) Mass increase of PLLA/Gel composite fiber incubated in SBF for 10 days. (d) Mass increase of PLLA/Gel/Ag composite fiber incubated in SBF for 10 days.
Fig. 5 BMSCs morphology and density cultured on the surface of composite fibers. SEM micrographs showing the morphology of BMSCs cultured on (a) PLLA, (b) PLLA/Gel and (c) PLLA/Gel/Ag surface for 7 days. (d) CCK-8 data plot showing the density of BMSCs cultured on each group of composite fibers for 1, 3, 5 and 7 days. (e) Fluorescence staining of BMSCs cultured on PLLA/Gel/Ag surface on day 7.
Fig. 6 VECs morphology and density cultured on the surface of composite fibers.(a) SEM micrographs of VECs cultured on composite fibers for 1, 3, 5 and 7 days.(b) CCK-8 data plot of VECs cultured on composite fibers for 1, 3, 5 and 7 days.
Fig. 7 (a) Qualitative analysis of the antibacterial property of composite fibers. (b) quantitative analysis of the antibacterial property of composite fibers.
Scheme 1. Construction mechanism of antibacterial, osteoinductive PLLA-based multifunctional fiber.
Fig. 1 The (a) morphology, (b) average diameter, (c) water contact angle and (d) surface energy of PLLA/Gel composite fiber.
Fig. 2 The (a) morphology, (b) average diameter, (c) water contact angle, (d) surface energy of PLLA/Gel /Ag composite fiber.
Fig. 3 The (a) TGA result and (b) Ag release profile of PLLA/Gel/Ag composite fiber.
Fig. 4 Mineralization of composite fibers. (a) The bioactivity of PLLA/Gel composite fiber (SEM). (b) The bioactivity of PLLA/Gel/Ag composite fiber (SEM). (c) Mass increase of PLLA/Gel composite fiber incubated in SBF for 10 days. (d) Mass increase of PLLA/Gel/Ag composite fiber incubated in SBF for 10 days.
Fig. 5 BMSCs morphology and density cultured on the surface of composite fibers. SEM micrographs showing the morphology of BMSCs cultured on (a) PLLA, (b) PLLA/Gel and (c) PLLA/Gel/Ag surface for 7 days. (d) CCK-8 data plot showing the density of BMSCs cultured on each group of composite fibers for 1, 3, 5 and 7 days. (e) Fluorescence staining of BMSCs cultured on PLLA/Gel/Ag surface on day 7.
Fig. 6 VECs morphology and density cultured on the surface of composite fibers.(a) SEM micrographs of VECs cultured on composite fibers for 1, 3, 5 and 7 days.(b) CCK-8 data plot of VECs cultured on composite fibers for 1, 3, 5 and 7 days.
Fig. 7 (a) Qualitative analysis of the antibacterial property of composite fibers. (b) quantitative analysis of the antibacterial property of composite fibers.
Highlights
² Biomacromolecules
fibers
for
improving
anti-infection
and
osseointegration. ² Silver nanoparticles acted as heterogeneous nucleation sites for calcium phosphorus. ² Multi-functional biomacromolecules composite fiber with good bioactivity. ² Improved adsorption capacity through hydrophilic groups of fiber.
Graphical Abstract
Silver nanoparticles were uniformly distributed on the inner of biomacromolecules composite fibers by co-electrospinning for improving anti-infection ability and osseointegration.
S0141-8130(19)36134-3 https://doi.org/10.1016/j.ijbiomac.2019.09.156 BIOMAC 13433
To appear in:
International Journal of Biological Macromolecules
Received Date: Revised Date: Accepted Date:
3 August 2019 19 September 2019 23 September 2019
Please cite this article as: X. Cheng, Q. Wei, Y. Ma, R. Shi, T. Chen, Y. Wang, C. Ma, Y. Lu, Antibacterial and osteoinductive biomacromolecules composite electrospun fiber, International Journal of Biological Macromolecules (2019), doi: https://doi.org/10.1016/j.ijbiomac.2019.09.156
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© 2019 Published by Elsevier B.V.
Antibacterial and osteoinductive biomacromolecules composite electrospun fiber Xuewei Cheng a#, Qin Wei b#, Yingao Ma a, Rui Shi a, Tongtong Chen c , Yingbo Wang a*, Chuang Ma d*, Yong Lu c* a
College of Chemical Engineering, Xinjiang Normal University, Urumqi, 830054,
Xinjiang, P. R. China. b
Animal Laboratory Center, Xinjiang Medical University, 393 Xinyi Road, Urumqi
830054, P. R. China. c
Radiology Department, Ruijin Hospital, Shanghai Jiao Tong University School of
Medicine, 197 Ruijin 2nd Road, Shanghai 200025, P. R. China. d
Department of Orthopedics Center, the First Affiliated Hospital of Xinjiang Medical
University, 393 Xinyi Road, Urumqi 830054, P. R. China.
#
These authors contributed equally.
* Corresponding Author: [email protected] [email protected] (Y. L).
(Y.
W);
[email protected]
(C.
M);
Abstract: Bone implant materials have been widely used in bone therapy. However, bone infections caused by bacteria can damage the healing and repairing ability of bone tissue, which remains a major problem to be solved in clinical bone regeneration with implants. In this study, silver nanoparticles (Ag-NPs) were uniformly distributed on the inner of Polylactic acid and Gelatin composite fibers (PLLA and Gel, biological macromolecules) by co-electrospinning for improving anti-infection ability and osseointegration. The optimized experimental conditions for this method were having PLLA/Gel mass ratio of 90:10 and Ag content of 7%. Ag-NPs acted as heterogeneous nucleation sites for promoting the nucleation and growth of calcium phosphorus on the surface of composite fiber. Bone marrow-derived mesenchymal stem cells adhered and proliferated well on the surface of the composite fiber, and the positive fluorescence staining indicated the existence of osteoblasts. Vascular endothelial cells had a good adherence and proliferation on the surface of composite fiber, showing good angiogenic properties. Antibacterial rate of the composite fiber was all over 97% against Monilia albicans, Escherichia coli, Staphylococcus aureus and
Pseudomonas
aeruginosa,
showing
good
antibacterial
property.
A
multi-functional biomacromolecules composite fiber was constructed and shown good bioactivity, osteoinductivity, angiogenic and antibacterial properties. Keywords: Electrospinning, composite fibers, biomacromolecules, osteoinductivity, anti-infection
1 Introduction Bone infection is a difficult problem needs to be overcome in clinical bone treatment [1] as it damages the self-healing ability of bone tissue, leading to bone loss and implantation failure [2]. Therefore, the construction of biomaterials that inhibit bone infection has important practical significance. Bone infections are usually caused by bacteria [3], and the most widely-used treatment methods for bone infections are debridement and systemic antibiotic therapy [4]. However, long-term and excessive antibiotic treatment may increase the risk of bacterial resistance [5], besides burst release issue of antibiotics after implantation [6, 7]. Therefore, reducing the bacterial resistance and improving the anti-infection ability of biomaterials has become an urgent need. Electrospinning can be used to construct fiber structures that are morphologically controllable and able to mimic natural extracellular matrix (ECM) [8]. Inspired by this, an ideal biomimetic environment can be designed for cell adhesion and reproduction to promote new tissue growth. Aurelija et al. [9] found that electrospinning can be used in the development of multilayer scaffolds with different biomacromolecules, in which polylactic acid (PLLA), fibrin, geltatin (Gel) and collagen are appropriate environment materials for cell transplantation and form the inner layer of the multilayer scaffold, while polyimide (PI) film can be used as a barrier to block cell migration from the scaffold. Hiroshi et al. [10] prepared submicron-sized polylactic acid/hydroxyapatite (PLLA/HA) fibers using electrospinning and the fibers showed promoted osteoblast distribution and proliferation in vitro. Shao et al. [11] prepared PLLA/tussah silk fibroin (PLLA/TSF) nanofibers by electrospinning, which possessed excellent mechanical properties and good biocompatibility. Pan et al. [12] incorporated Fe3O4 nanoparticles into PLLA fibers by electrospinning and the composite fibers showed high-level bone healing activity. However, the related studies did not consider infection issues during bone regeneration, which is an urgent problem to be solved in as early as the design and synthesis stage of electrospun bone-like fibers.
PLLA and its composites with inorganic materials have been applied and investigated thoroughly in the field of orthopedics[13, 14] . Johnson et al. [15] mixed nanometer hydroxyapatite (n-HA) with PLLA and polycaprolactone (PCL) and deposited them as nanometer coatings on the surface of magnesium matrix, which effectively reduced the degradation rate in vivo, promoted bone formation and guided bone regeneration, but its antibacterial property was not enough to meet clinical requirements. The long-term antibacterial function of biomaterials has always been highly valued by researchers [16]. Silver nanoparticles (Ag-NPs) have a broad antibacterial spectrum that kills up to hundreds of pathogenic bacteria and viruses [17]. Xu et al. [18]deposited a mixed nano-film composed of tannic acid (TA) and Fe3+ on a Ti substrate, and Ag-NPs was wrapped in the nano-film. They found that Ag-NPs were easily aggregated, reducing its antibacterial activity [19]. Gao et al. [20] added Ag-NPs into PLLA fibers using electrospinning and their studies showed that Ag-NPs formed heterogeneous nucleation sites and improved the bioactivity of the material surface. Yang et al. [21]synthesized PLLA fiber membrane by electrospinning, and uniformly distributed Ag-NPs on the surface of fiber membrane by Michael addition reaction. The study showed that the fiber had good antibacterial property. Therefore, the construction of bioactive and osteoinductive PLLA-based biomacromolecules composite fibers with uniform distribution of Ag-NPs, antibacterial and anti-infective properties is an urgent need. Based on the outstanding broad-spectrum antibacterial property of Ag-NPs, this study has successfully constructed biomacromolecules-based antibacterial and osteoinductive composite fibers with uniformly distributed Ag-NPs on the inner surface of the fibers by electrospinning. Firstly, Ag-NPs can attract bone marrow-derived mesenchymal stem cells (BMSCs) from the surrounding area of the implant to the fracture site [22], and they can enter BMSCs, bind to DNA, activate the expression of genes such as HIF-1α and IL-8 and promote cell proliferation. Besides, Ag-NPs can activate TGF-β/BMP signaling pathway[23, 24], induce osteogenic differentiation of BMSCs and activate endothelial nitric oxide synthase (eNOS) to generate NO free radicals by serine phosphorylation[25]. NO contributes to the
mitogenic effect of several growth factors such as vascular endothelial growth factor (VEGF), enhance the proliferation and migration of endothelial cells and controls angiogenesis[26]. In addition, Ag-NPs have a high reduction potential of 0.7996 V, which can destroy the free energy of bacterial transmembrane proton kinetic storage (proton motive force, PMF)[27] and affect the metabolic reaction of bacteria. The level of reactive oxygen species (ROS) is thus rapidly raised up during the above process, break the steady state of ROS in bacteria and cause them to rupture and die. The synthesis mechanism of antibacterial and osteoinductive PLLA-based multifunctional fiber is shown in Scheme 1.
Scheme 1. Construction mechanism of antibacterial, osteoinductive PLLA-based multifunctional fiber.
2 Materials and methods 2.1 Materials PLLA was purchased from Jinan Daigang Biomaterial Co., Ltd., China. Silver nanoparticles (Ag-NPs) with diameter of less than 100 nm was purchased from
Sigma-Aldrich.
Gelatin
(Gel)
was
from
Beijing
Chemical
Plant,
China.
Trifluoroethanol (TFE) was from Sigma-Aldrich, US. 2.2 Test instruments TL01 Electrospinning Machine was from Shenzhen Tongli Micro-Nano Technology Co., Ltd., China. Scanning electron microscope (SEM, LEO-1430VP) was from Carl Zeiss, Germany. Water contact angle analysis instrument (SDC-200) was from Dongguan Sindin Precision Instrument Co., Ltd., China. Thermogravimetric analysis (TGA) instrument (STA 449F3, heating rate at 3 K·min-1, temperature ranging from 10 °C ~1000 °C) was from NETZSCH, Germany. Atomic Absorption Spectrometer (AAS, Z-2000) was from Hitachi, Japan. Confocal microscope (ECLIPSE Ti) was from Nikon, Japan and microplate reader was from Thermo Fisher Scientific, US. 2.3 Preparation of PLLA/Gel/Ag composite fiber Gel weighing 0.16 g was dissolved in 9 mL of TFE and stirred thoroughly until Gel was completely dissolved. Then 1.44 g of PLLA and various amount of Ag-NPs were added to the solution and stirred thoroughly, configured into electrospinning suspensions with Ag content of 3%, 5%, 7% and 9%. The suspensions were then transferred to 20 mL plastic syringes equipped with medical-grade 6# stainless steel needles, adjusted using a syringe pump, and a high voltage of 18 kV·cm-1 was supplied through a high-voltage DC power source at a constant sampling rate of 3 mL·h-1. Electrospinning fibers were collected on aluminum foil-coated stainless steel rotating mandrel (20 cm × 13 cm) placed 15 cm away from the needle. 2.4 Ion release test Composite fiber was soaked in 40 mL of phosphate buffered saline (PBS, pH=7.4) and incubated at 37 °C for 10 days to test Ag+ release profile. The absorbance of Ag+ in the leachate was measured by AAS (table S1) and Ag+ concentration was calculated from its standard curve, in order to evaluate the physiological stability of the composite fiber.
2.5 Bioactivity test Composite fibers were cut into squares of 20 mm × 20 mm, weighed and placed in 37 °C, 40 mL, 2× simulated body fluid (SBF, table S2) for mineralization (n = 3 for each experimental group). SBF was replaced every 24 hours. Samples were taken out at different mineralization time points (1, 3, 5, 7, 10 days) and soaked in 400 mL of deionized water overnight to remove soluble inorganic ions. Samples were then dried at room temperature for 72 hours, weighed again and spray coated with gold to increase surface conductivity, followed with SEM characterization to observe the surface morphology of the samples. The mineralization quantity of calcium and phosphorus salt on the fiber surface was determined by the difference of fiber mass before and after mineralization. 2.6 In vitro evaluation of primary bone marrow-derived mesenchymal stem cells Bone marrow-derived mesenchymal stem cells (BMSCs) were harvested from the bone marrow of 10 days old SD rats. All animal experiment procedures were carried out in accordance with the Guide for the Care and Use of Laboratory Animals and all animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC) of the First Affiliated Hospital of Xinjiang Medical University. Cell study consisted of 3 experimental groups, including PLLA, PLLA/Gel and PLLA/Gel/Ag (n = 7 for each experimental group). The samples were placed in a culture plate, and BMSCs were seeded on the surface of the samples at a seeding density of 5×104 cell·mL-1 with 3.0 mL of α-MEM medium added to each well, and cultured at 37 °C in a CO2 incubator. After 1, 3, 5, and 7 days of culture, the samples were washed with PBS for three times and followed with fixation and dehydration. Then samples were dried and coated with gold to observe the morphology of the cells. CCK-8 was used to examine cell proliferation at the time point of 1, 3, 5, and 7 days. Briefly, 100 μL of cell suspension was obtained per well (n = 3 per sample), mixed with 10 μL of CCK-8 in a 96-well plate and incubated at 37 °C in a CO2 incubator for 3 hours. The absorbance of each well was obtained by a microplate reader at a wavelength of 450 nm. In addition, FITC staining was performed on the cells after 7
days of culture. Cells were first fixed with paraformaldehyde, then treated with Triton X-100 and blocked with goat serum for 20 min. After that, cells were incubated with FITC at 37 °C for 1.5 hours, and counterstained with DAPI for 5 minutes. Each of the above procedures was followed with three times of PBS wash before the next step. 2.7 In vitro evaluation of primary vascular endothelial cells Vascular endothelial cells (VECs) were harvested from the kidney of newborn (3 days old) SD rats. All animal experiment procedures were carried out in accordance with the Guide for the Care and Use of Laboratory Animals and all animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC) of the First Affiliated Hospital of Xinjiang Medical University. Cell study consisted of 3 experimental groups, including PLLA, PLLA/Gel and PLLA/Gel/Ag (n = 7 for each experimental group). The samples were placed in a culture plate, and VECs were seeded on the surface of the samples at a seeding density of 1.5×104 cell·mL-1 with 3.0 mL of α-MEM medium added to each well, and cultured at 37 °C in a CO2 incubator. After 1, 3, 5, and 7 days of culture, samples were washed with PBS for three times, fixed and dehydrated, dried and coated with gold to observe the morphology of the cells. CCK-8 was used to examine cell proliferation at each time point with the similar procedure as samples cultured with BMSCs. 2.8 Antibacterial property test The strains used for antibacterial test were Monilia albicans (M. albicans), Escherichia coli (E. coli), Staphylococcus aureus (S. aureus) and Pseudomonas aeruginosa (P. aeruginosa). All strains were tested in three experimental groups, including PLLA/HA, PLLA/Gel and PLLA/Gel/Ag (n = 3 for each experimental group). For the qualitative analysis, stains of M. albicans, E. coli, S. aureus and P. aeruginosa were inoculated on MH medium separately for activation culture. Samples of each strain were acquired and suspended in separate physiological saline to prepare 10 mL bacterial suspensions with a concentration of 0.5 McFarland unit (0.5 MCF, 1.5×108 cell·mL-1). Then 50 mL of each suspension was obtained, seeded
on a plate and cultured at 37 °C in an incubator for 24 hours. For the quantitative analysis, the strains were inoculated on MH medium for three times to obtain purified colonies. The samples to be tested were sterilized with ultraviolet light for 1 hour before transferred to a glass slide in a Petri dish. In order to prevent the evaporation of the bacterial suspension, sterile water was added to cover the bottom of the Petri dish. Single bacteria colonies were scraped off with inoculating loop and prepared into suspension of 0.5 MCF using physiological saline. 50 μL of the as prepared bacterial suspension was dropped onto the surface of the sample, and cultured in a 37 °C incubator for 24 hours. Afterwards, 20 μL of the remaining bacterial suspension on the surface of the sample was obtained and seeded on a plate, and the number of colonies was counted after another 24 hours of culture. The antibacterial property of the material was represented by antibacterial rate. Three parallel experiments were performed to calculate the average antibacterial rate. The calculation method is shown in equation (1), ݁ݐܽݎ݈ܽ݅ݎ݁ݐܾܿܽ݅ݐ݊ܣൌ
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ൈ ͳͲͲΨ
˄1˅
where CCCG is the colony count of the control group and CCEG is the colony count of the experimental group. 2.9 Statistical analysis A One Sample t Test (assuming unequal variance) was used for statistical analysis and all data are presented as mean ± standard deviation. The difference between two sets of data is considered statistically significant when p < 0.05.
3 Results and discussions 3.1 Preparation of composite fibers Fig. 1 shows the morphology, average diameter, contact angle, surface energy, weight loss and Ag+ release profile of the biomacromolecules composite fibers. It can be seen from Fig. 1a that when the mass ratio of PLLA/Gel was 100/0, the fiber
morphology was inconsistent and fibers were relatively thick. While with the addition of Gel and the mass ratio of PLLA/Gel changed to 90/10 and 80/20, the fiber morphology became relatively homogeneous. However, beads appeared in the fibers when the mass ratio of PLLA/Gel changed to 70/30 (Fig. S1). This was because the addition of Gel has increased the viscosity of the electrospinning solution, causing a continuous increase of surface tension that the electric field force was insufficient to overcome, resulting in uneven fiber diameters during electrospinning process. The average diameters of the PLLA/Gel composite fibers prepared at mass ratio of 100/0, 90/10, 80/20 and 70/30 were 909 nm, 373 nm, 592 nm and 246 nm, respectively (Fig. 1b). Studies have shown that the addition of Gel could increase surface contact angle and thus improving the hydrophilicity of the composite materials due to the rich content of hydroxyl and hydrophilic groups in Gel[28]. According to the water contact angle data of PLLA/Gel (Fig. 1c), the surface contact angle of the composite fiber was 139.2°, 144.1°, 135.9°, and 138.2° with the addition of Gel. According to literature report[29], the hydrophobicity of electrospun fibers increase due to special fiber structure, and the size of the fiber also affects the hydrophobicity of the material. When Gel was added to pure PLLA fiber, the average fiber diameter decreased from 909 nm to 373 nm, and the water contact angle of 90/10 composite fiber was slightly larger than that of pure PLLA fiber. With the increase of Gel addition, the surface water contact angle of 80/20 composite fiber was slightly small than 90/10 group, indicating that the addition of Gel had an effect on the water contact angle, but the effect was not significant[30]. However, when PLLA/Gel mass ratio increased to 70/30, the water contact angle of the composite fiber increased slightly when compared to 80/20 composite fiber, which was due to the smallest diameter of the 70/30 composite fiber. The above results were consistent with Wenzel's theory that the combination of low surface energy and high surface roughness produces a hydrophobic surface (Fig. 1d)[31].
Fig. 1 The (a) morphology, (b) average diameter, (c) water contact angle and (d) surface energy of PLLA/Gel composite fiber.
As Ag is considered to be an ideal inorganic antibacterial agent and able to promote the growth and proliferation of BMSCs [32], Ag-NPs were added to PLLA/Gel (90/10) fibers to construct an antibacterial composite fiber. The surface of the composite fiber with different content of Ag-NPs was smooth, and Ag-NPs were evenly distributed in the fiber. It can be seen from Fig. 2a that no droplets were found on the surface of 3%, 5% and 7% Ag-NP-containing fibers and the diameter of the fibers decreased but the distribution was relatively uniform with the increase of Ag-NPs content. However, the fibers became disordered and easily broken when Ag-NPs content increased to 9% (Fig. S2). This was because the high concentration of Ag-NPs resulted in a low viscosity of the electrospinning liquid, and the electrical conductivity of Ag-NPs made fibers easily stretched discontinuously to break during the electrospinning process. The average diameter of PLLA/Gel/Ag composite fiber (Fig. 2b) was 389 nm, 326 nm, 282 nm and 328 nm for Ag-NPs content of 3%, 5%, 7% and 9%, respectively. The average diameter of the composite fiber decreased along with the increase of Ag-NPs content from 3% to 7%. This was caused by the dropped viscosity of the electrospinning liquid with continues addition of Ag-NPs, the solvent volatilization was accelerated, and the surface tension of the fiber was reduced to easily get stretched under the same electric field force. When the content of Ag-NPs
increased to 9%, a discontinuous and uneven distribution of fibers lead to an increase in the average diameter of the biomacromolecules composite fibers. The hydrophilicity test of the fibers (Fig. 2c) showed that the water contact angles were 143.3°, 144.3°, 138.3° and 138.5° with increasing Ag-NPs content of 3%, 5%, 7% and 9%, respectively. It can be seen from the test result that the water contact angle of composite fibers increased slightly when Ag-NPs content increased from 3% to 5% and then decreased when Ag-NPs content increased to 7%. When Ag-NPs content continued to increase to 9%, the fibers were discontinuous and unevenly distributed, with slightly increased average diameter and water contact angle. The surface energy test of the composite fibers showed that the surface energy of the fiber with 7% Ag-NPs content was the highest (Fig. 2d). From the above characterizations including morphology, average diameter and hydrophilicity of the composite fibers, it was known that the fiber with 7% Ag-NPs addition was the optimum group in this study. The tested Ag-NPs content was 4.4% compared to the designated 7% in the composite fiber (Fig. 3a), and the Ag+ release was stable (Fig. 3b).
Fig. 2 The (a) morphology, (b) average diameter, (c) water contact angle, (d) surface energy of PLLA/Gel /Ag composite fiber.
Fig. 3 The (a) TGA result and (b) Ag release profile of PLLA/Gel/Ag composite fiber.
3.2 Bioactivity of the composite fibers Bioactivity is an important criterion of materials for the integration and repair of human bone tissue. Fig. 4 is a graph showing the bioactivity of the biomacromolecules composite fiber surface after soaked in SBF for mineralization. It can be seen from Fig. 4a that when the PLLA/Gel mass ratio was 100/0, the surface of the fiber was partially coated with nanoparticles after 10 days of mineralization, and the nanoparticles had a tendency to join into pieces. When the mass ratio of PLLA/Gel was 90/10, the surface of the biomacromolecules composite fiber was all covered with nanoparticles. However, the surface of the composite fiber with PLLA/Gel mass ratio of 80/20 had less calcium and phosphorus salt deposition and almost no deposition on the surface of PLLA/Gel fibers with mass ratio of 70/30. With the increase of soaking time, the deposition amount of the nanoparticles on PLLA/Gel fiber surface has increased (Fig. 4c). On the first 5 days, there was not much increase in the deposition of calcium and phosphorus salt on the fiber surface, but difference showed up after 5 days of mineralization, and the deposition of 90/10 composite fiber was the highest. Studies have shown that, the more the Gel content, the stronger the hydrophilicity of the biomacromolecules composite fiber, which was not advantageous for the deposition of calcium and phosphorus salt on the fiber surface. Therefore, the composite fiber with PLLA/Gel mass ratio of 90/10 was the best in this study. It can be seen in Fig. 4b that after 10 days of mineralization, there was no calcium and phosphorus salt deposition on the fiber surface with 3% Ag-NPs addition,
and only small amount of deposition on the fiber surface with 5% Ag-NPs addition. The calcium and phosphorus salt deposition was the highest on the fiber surface with 7% Ag-NPs addition and the fibers were wrapped all over by salt to form a uniform spread. However, the calcium and phosphorus salt only deposited on the surface of the fiber with 9% Ag-NPs addition but not covered the whole surface. Overall, the salt deposition quantity increased with the expanded soaking time, and decreased with the enlarged fiber diameter, among which the deposition of calcium and phosphorus salt reached the maximum on the surface of composite fiber with 7% Ag-NPs addition on day 10 (Fig. 4d). The mineralization mechanism was analyzed accordingly. First of all, the fiber surface had interactions with SBF at the initial stage of soaking and PLLA hydrolyzed some functional groups to produce carboxyl groups. Secondly, the carboxyl groups on the surface of the fiber attracted Ca2+ to form calcium and phosphorus salt granules, which acted as nucleation sites to form a coarse calcium and phosphorus salt layer as the soaking time increased, providing a larger nucleation space for the further deposition of calcium and phosphorus salt. In the meanwhile, Ag-NPs acted as heterogeneous nucleation sites and aided the increase of deposition quantity of calcium and phosphorus salt on the surface of composite fibers. Therefore, the deposition of calcium and phosphorus salt on the surface of composite fibers was the result of the synergistic effect of heterogeneous nucleation of Ag-NPs and composite fiber diameter[19]. The above analysis
demonstrated that the
PLLA/Gel/Ag composite fiber with 7% Ag-NPs addition had the best bioactivity. In summary, the optimal experimental conditions were PLLA/Gel mass ratio of 90/10 and Ag-NPs content of 7%. The following experiments including in vitro cell culture and antibacterial test of the composite fibers were conducted under these conditions.
Fig. 4 Mineralization of composite fibers. (a) The bioactivity of PLLA/Gel composite fiber (SEM). (b) The bioactivity of PLLA/Gel/Ag composite fiber (SEM). (c) Mass increase of PLLA/Gel composite fiber incubated in SBF for 10 days. (d) Mass increase of PLLA/Gel/Ag composite fiber incubated in SBF for 10 days.
3.3 In vitro cell compatibility of composite fibers Fig. 5 shows the morphology, cell viability and fluorescence staining of BMSCs cultured on the surface of composite fibers for 7 days. The CCK-8 result (Fig. 5d) showed that cells adhered well on the surface of different materials and cell densities were close after inoculated on the surface of the fibers for one day. After 3 days, the cells were adapted to the surrounding environment of the material and began to grow. When the cells were cultured for 5 days, they began to proliferate and BMSCs had the best cell activity on the PLLA/Gel/Ag fiber surface when cultured for 7 days. Besides, BMSCs showed a spread morphology on the surface of PLLA, PLLA/Gel and PLLA/Gel/Ag composite fibers on day 7 (Fig. 5a, b) and BMSCs density was the highest on the surface of PLLA/Gel/Ag composite fibers (Fig. 5c). The fluorescence staining of BMSCs on PLLA/Gel/Ag composite fibers was positive (Fig. 5e), which indicated the differentiation of BMSCs into
osteoblasts. Studies have shown that Ag-NPs could promote the proliferation and differentiation of BMSCs, therefore, PLLA/Gel/Ag composite fibers could also promote the proliferation of BMSCs and induce its osteogenesis, showing good osteoinductivity. The mechanism of inducing osteogenesis was that after the cells adhered to the surface of the composite fibers, Ag-NPs attracted the MSCs around the implant to the fracture site and entered MSCs[22], bind to DNA and activated gene expression of HIF-1α and IL-8. This process has promoted cell proliferation, and Ag-NPs could induce and activate TGF-β/BMP signaling pathway, inducing osteogenic differentiation of MSCs[23, 24].
Fig. 5 BMSCs morphology and density cultured on the surface of composite fibers. SEM micrographs showing the morphology of BMSCs cultured on (a) PLLA, (b) PLLA/Gel and (c) PLLA/Gel/Ag surface for 7 days. (d) CCK-8 data plot showing the density of BMSCs cultured on each group of composite fibers for 1, 3, 5 and 7 days. (e) Fluorescence staining of BMSCs cultured on PLLA/Gel/Ag surface on day 7.
The human bone contains a large number of microvessels for the transport of nutrients, so the cytocompatibility of composite fibers with VECs were tested in this study. Fig. 6a is an SEM micrograph of VECs cultured on the surface of composite fibers for 1, 3, 5, and 7 days. The micrograph showed that VECs began to adhere to the surface of the composite fiber, and their pseudopods were extended after 1 and 3 days of culture. Then after 5 and 7 days, VECs began to proliferate and spread on the surface of the composite fiber. Cell activity was tested using CCK-8 and the
result showed (Fig. 6b) that as the culture time continued to increase, the number and activity of VECs has increased. Cell activity was the highest on PLLA/Gel/Ag surface at different culture time points. This was because that a certain concentration of Ag-NPs could stimulate the production of NO by eNOS in cells, and NO could contribute to the mitogenic effect of several growth factors such as VEGF, beneficial for
vascularization[25,
26].
PLLA/Gel/Ag
composite
fiber
has
good
cytocompatibility with VECs and it can promote the growth as well as proliferation of VECs, inducing angiogenesis.
Fig. 6 VECs morphology and density cultured on the surface of composite fibers.(a) SEM micrographs of VECs cultured on composite fibers for 1, 3, 5 and 7 days.(b) CCK-8 data plot of VECs cultured on composite fibers for 1, 3, 5 and 7 days.
3.4 Antibacterial property of composite fibers Gram-negative and Gram-positive bacteria that are commonly seen in clinical treatment, including M. albicans, E. coli, S. aureus, and P. aeruginosa were used in this antibacterial study. It can be seen from Fig. 7a that after 24 hours of incubation, E.
coli and P. aeruginosa on the surface of PLLA fiber were undergoing strong metabolism and reproduction activity, while S. aureus and P. aeruginosa were more active on reproduction on the surface of PLLA/Gel. There were almost no colonies in the medium of the PLLA/Gel/Ag group and that was because the addition of Ag-NPs has enhanced the antibacterial property of the composite fiber. Quantitative analysis (Fig. 7b) showed that the antibacterial rates of PLLA against M. albicans and S. aureus were 80.7% and 74.2%, respectively, and there was no antibacterial activity against E. coli and P. aeruginosa. PLLA/Gel had an antibacterial rate of 90.6% against E. coli but no antibacterial ability against all the other three bacteria strains. With the addition of Ag-NPs, PLLA/Gel/Ag had antibacterial rates over 97% for all four strains. The results showed that PLLA/Gel/Ag composite fiber has good antibacterial ability against E. coli, S. aureus, P. aeruginosa and M. albicans. The main mechanism of the antibacterial activity of Ag-NPs is the metal ion effect and catalysis action. First of all, the cell membrane of Gram-positive and Gram-negative bacteria is negatively charged[33], and it is easy to attract positively charged Ag+ due to the action of static electricity, inactivate cell enzymes and destroy the integrity of cell membrane. In addition, Ag+ interacts with thiol groups in proteins to promote the release of oxygen-active substances, which can cause damage to proteins and DNA, leading to cell death[34]. Secondly, the chemical structure of Ag-NPs provides a strong catalytic ability and a high reduction potential, resulting in the production of ROS in cells, causing bacteria death by strong oxidizing properties[32]. Besides, bacteria use PMF, established by a proton pump, to produce adenosine triphosphate (ATP) through ATP synthase. However, Ag-NPs can destroy PMF, oppress and even disrupt the metabolic reactions of bacterial cells, rapidly increase the level of ROS in bacteria and thus cause death[27]. In addition, Ag-NPs can migrate out of dead bacteria and repeat the above cycle, which is why the antibacterial activity of Ag-NPs is significantly sustainable. Although the membrane structure of Gram-positive and Gram-negative bacteria is different, most bacteria are negatively charged. Therefore, Ag+ can cause bacteria death by electrostatic attraction. Literature has reported that the antibacterial activity of Ag-NPs may be related to the
characteristics of the bacterial species[35]. The outer cell of Gram-negative bacteria has a layer of lipopolysaccharide, followed by a thin layer of peptidoglycan, while the cell wall of Gram-positive bacteria is mainly composed of a thick layer of peptidoglycan. Studies have shown that Ag-NPs have better antibacterial properties against Gram-negative bacteria.
Fig. 7 (a) Qualitative analysis of the antibacterial property of composite fibers. (b) quantitative analysis of the antibacterial property of composite fibers.
4 Conclusions This study has successfully prepared PLLA/Gel/Ag composite fibers with good bioactivity,
osteoinductivity,
angiogenicity
and
anti-infective
property
by
electrospinning on the basis of rich hydrophilic groups in Gel and antibacterial property of Ag-NPs. The results showed that a uniform morphology of the biomacromolecules composite fiber was obtained when the mass ratio of PLLA to Gel
was 90:10 and the content of Ag-NPs was 7%. The rich hydrophilic groups -OH and -NH2 in Gel has improved the adsorption capacity of the composite fiber, and Ag-NPs acted as heterogeneous nucleation sites, promoted the nucleation and growth of calcium and phosphorus salt on the surface of the fiber, and improved the bioactivity of the fiber together with Gel, showing synergistic function. BMSCs adhered and proliferated well on the surface of PLLA/Gel/Ag composite fibers, and the fluorescence staining of cells was positive, indicating osteogenesis of BMSCs. VECs also adhered and proliferated well on the surface of the composite fiber, indicating good angiogenic property. Besides, the antibacterial rate of PLLA/Gel/Ag composite fiber was as high as over 97% against all four strains including M. albicans, E. coli, S. aureus and P. aeruginosa. In this study, electrospinning technology was used to successfully construct multifunctional composite fibers with good bioactivity, osteoinductivity, angiogenic and antibacterial properties. Therefore, PLLA/Gel/Ag composite fibers are potentially viable bone tissue repair materials that can inhibit bone infection and promote osseointegration.
Acknowledgments This work was supported by the National Natural Science Foundation of China (51662038, 81560350 and 81760397), Outstanding Youth Natural Science Foundation Project of Xinjiang (2017Q002), Research and Innovation Project of Postgraduates in Autonomous Region at 2019. Xinjiang Normal University 2018 College Students Innovative
and
Entrepreneurship
Training
Project
(201810762007
and
201810762013), Supported by the “13th Five-Year” Plan for Key Discipline Chemistry, Xinjiang Normal University.
Competing interests We declare that we have no conflicts of interest.
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Figure captions Scheme 1. Construction mechanism of antibacterial, osteoinductive PLLA-based multifunctional fiber.
Fig. 1 The (a) morphology, (b) average diameter, (c) water contact angle and (d) surface energy of PLLA/Gel composite fiber.
Fig. 2 The (a) morphology, (b) average diameter, (c) water contact angle, (d) surface energy of PLLA/Gel /Ag composite fiber.
Fig. 3 The (a) TGA result and (b) Ag release profile of PLLA/Gel/Ag composite fiber.
Fig. 4 Mineralization of composite fibers. (a) The bioactivity of PLLA/Gel composite fiber (SEM). (b) The bioactivity of PLLA/Gel/Ag composite fiber (SEM). (c) Mass increase of PLLA/Gel composite fiber incubated in SBF for 10 days. (d) Mass increase of PLLA/Gel/Ag composite fiber incubated in SBF for 10 days.
Fig. 5 BMSCs morphology and density cultured on the surface of composite fibers. SEM micrographs showing the morphology of BMSCs cultured on (a) PLLA, (b) PLLA/Gel and (c) PLLA/Gel/Ag surface for 7 days. (d) CCK-8 data plot showing the density of BMSCs cultured on each group of composite fibers for 1, 3, 5 and 7 days. (e) Fluorescence staining of BMSCs cultured on PLLA/Gel/Ag surface on day 7.
Fig. 6 VECs morphology and density cultured on the surface of composite fibers.(a) SEM micrographs of VECs cultured on composite fibers for 1, 3, 5 and 7 days.(b) CCK-8 data plot of VECs cultured on composite fibers for 1, 3, 5 and 7 days.
Fig. 7 (a) Qualitative analysis of the antibacterial property of composite fibers. (b) quantitative analysis of the antibacterial property of composite fibers.
Scheme 1. Construction mechanism of antibacterial, osteoinductive PLLA-based multifunctional fiber.
Fig. 1 The (a) morphology, (b) average diameter, (c) water contact angle and (d) surface energy of PLLA/Gel composite fiber.
Fig. 2 The (a) morphology, (b) average diameter, (c) water contact angle, (d) surface energy of PLLA/Gel /Ag composite fiber.
Fig. 3 The (a) TGA result and (b) Ag release profile of PLLA/Gel/Ag composite fiber.
Fig. 4 Mineralization of composite fibers. (a) The bioactivity of PLLA/Gel composite fiber (SEM). (b) The bioactivity of PLLA/Gel/Ag composite fiber (SEM). (c) Mass increase of PLLA/Gel composite fiber incubated in SBF for 10 days. (d) Mass increase of PLLA/Gel/Ag composite fiber incubated in SBF for 10 days.
Fig. 5 BMSCs morphology and density cultured on the surface of composite fibers. SEM micrographs showing the morphology of BMSCs cultured on (a) PLLA, (b) PLLA/Gel and (c) PLLA/Gel/Ag surface for 7 days. (d) CCK-8 data plot showing the density of BMSCs cultured on each group of composite fibers for 1, 3, 5 and 7 days. (e) Fluorescence staining of BMSCs cultured on PLLA/Gel/Ag surface on day 7.
Fig. 6 VECs morphology and density cultured on the surface of composite fibers.(a) SEM micrographs of VECs cultured on composite fibers for 1, 3, 5 and 7 days.(b) CCK-8 data plot of VECs cultured on composite fibers for 1, 3, 5 and 7 days.
Fig. 7 (a) Qualitative analysis of the antibacterial property of composite fibers. (b) quantitative analysis of the antibacterial property of composite fibers.
Highlights
² Biomacromolecules
fibers
for
improving
anti-infection
and
osseointegration. ² Silver nanoparticles acted as heterogeneous nucleation sites for calcium phosphorus. ² Multi-functional biomacromolecules composite fiber with good bioactivity. ² Improved adsorption capacity through hydrophilic groups of fiber.
Graphical Abstract
Silver nanoparticles were uniformly distributed on the inner of biomacromolecules composite fibers by co-electrospinning for improving anti-infection ability and osseointegration.