Electrospinning Fibers Modified with Near Infrared Light‐Excited Copper Nanoparticles for Antibacterial and Bone Regeneration

Bone infection is an inflammatory bone disease caused by infectious microorganisms, which can lead to progressive bone destruction and loss. It is still an urgent and unmet clinical need to develop a rapid and effective sterilized method to reduce bone infection. In this study, near‐infrared (NIR) light‐responsive polydopamine (PDA) adherent Copper nanoparticles (Cu‐NPs) are constructed using electrospun poly lactic acid (PLLA) fibers as a substrate (PLLA@PDA/Cu). Dopamine (DA) is self‐polymerized to synthesize the PDA on the fiber surface, which can reduce Cu2+ to form Cu‐NPs. Results show that the addition of PDA and Cu2+ significantly improves the hydrophilicity and bioactivity of the prepared PLLA@PDA/Cu composite fibers, demonstrating superior physiological stability. Due to the unique photothermal properties of Cu‐NPs, PLLA@PDA/Cu is able to generate a large amount of reactive oxygen species under 808 nm NIR laser irradiation, with a photothermal conversion efficiency of 23.7%. The antimicrobial effect of Cu‐NPs is synergistic with their photothermal effect, which potentiated the antibacterial rate of composite fibers . The results of cell experiments show that this composite fiber demonstrates satisfactory osteogenic and angiogenic properties. In conclusion, a photothermal antibacterial PLLA@PDA/Cu composite fiber with great potential in healing infectious bone defects is successfully engineered.


Introduction
Bone infection is still a major clinical challenge, in which bacteria can remain dormant within osteoblasts to evade the immune antibacterial ability, and a single function of PLLA [8] limit their clinical application.Therefore, the preparation of PLLA-based fibers with antibacterial and osteoinductive capabilities is an urgent and unmet clinical problem.
The antibacterial and osteoinductive properties of PLLA can be effectively improved by doping metal ions, broadening its application in the field of biomedicine. [9]It has been reported that Cu 2+ induced the formation of a hypoxic microenvironment, which increased the expression of Annexin2-S100A10 complex in osteoblasts.The upregulated expression of Annexin2-S100A10 complex, which contains a Ca 2+ binding site, further induced the increase of intracellular Ca 2+ . [10]Moreover, Cu 2+ affected the expression of several osteogenic-specific proteins, including osteocalcin (OCN) and bone morphogenetic protein-2 (BMP-2), thereby promoting osteogenesis. [11]As a trace element in the human body, Cu exists mainly as Cu 2+ , which has broad-spectrum antimicrobial properties. [12]Copper nanoparticle (Cu-NPs) are responsive to near-infrared (NIR) light and have strong photothermal properties. [13]The local high temperature generated by Cu-NPs under NIR irradiation can be utilized to treat bacterial infections.In addition, copper photothermal agents (PTA) also demonstrated the advantages of high controllability and mild invasiveness. [14]Chen et al. [15] incorporated Cu-NPs into the hydrogel and found that after irradiating the hydrogel with a laser for 10 min, a significant increase in temperature was observed, and the photothermal conversion rate reached 22.3%.The hydrogel exhibited photodynamic sterilization properties with an antibacterial rate against Escherichia coli (E.coli) to 79% after irradiation, Liu et al [16] prepared composite fibers PLLA/Dex/Ag modified with silver nanoparticle (Ag-NPs) by electrostatic spinning, the photothermal conversion efficiency was 10.3%, in addition, the photothermal conversion efficiency of Cu-NPs was higher than that of Ag-NPs, indicating that Cu-NPs as PTA had great potential in photodynamic sterilization.Therefore, it is promising to construct Cu-NPs with both antibacterial and osteoinductive properties on the surface of PLLA fibers.
In the current method for coating Cu-NPs on the surface of PLLA fiber, Leron et al. [17] used a liquid induced phase separation technique to prepare this PLLA film.The polydopamine (PDA) with mussel structure was applied on the surface of PLLA fiber as adhesion, and the Cu-NPs were established by reducing Cu 2+ to Cu-NPs.However, in this way, the prepared PLLA film had no pores and could not mimic the structure of the extracellular matrix, nor could it provide structural and biochemical support for cells and tissues. [18]Though the antibacterial rate reached 100% after 24 h of incubation with Bacillus subtilis, this elongated antibacterial time can easily lead to dysbiosis induced secondary infection. [19]Therefore, developing a powerful antibacterial osteoinductive and the Cu-NPs were established by reducing Cu 2+ to Cu-NPs for PLLA fiber is urgently needed.
In this study, combining the structure advantage of PLLA fibers to mimic the extracellular matrix and the potential photothermal antibacterial effects of Cu-NPs, a PDA-adherent and the Cu-NPs were established by reducing Cu 2+ to Cu-NPs with NIR responsive properties were self-assembled on the surface of electrospun PLLA fibers (PLLA@PDA/Cu).This modified PLLA@PDA/Cu fiber could achieve fast and potent antibacterial activity and promote bone regeneration.We characterized the preparation of composite fibers, and investigated their physio-logical stability, bioactivity, photothermal properties and antibacterial properties after light exposure, respectively.The endothelial cells were cultured in vitro to examine the cell morphology and metabolic activity, and the expression of hypoxia-inducible factor (HIF-1) and vascular endothelial growth factor (VEGF) on the 7th day to analyze the cause of osteogenesis.To further explore the osteogenic differentiation ability of the composite fiber on osteoblasts, alkaline phosphatase (ALP) and alizarin red (ARS) stainings were performed.Eventually, the in vivo cytotoxicity of the composite fiber was estimated via the tissue stainings.

Characterization of Composite Fibers
PLLA@PDA/Cu composite fibers were constructed by a combination of the electrospinning and self-assembly techniques.First, at the beginning of the dopamine (DA) polymerization process, DA was oxidized to dopamine quinone, which self-polymerized to form PDA. Second, after the addition of Cu 2+ , it chelated with the catechol group of PDA and was reduced to form Cu-NPs.In this way, Cu-NPs were stably and slowly released, and achieved long-acting antibacterial activities. [22]Then, after irradiating with NIR, the photothermal conversion properties of PDA and Cu-NPs induced a rise in local temperature, which lead to bacterial membrane destruction and protein denaturation. [23]Furthermore, the simultaneously generated reactive oxygen species (ROS) can also exert anti-bacterial functions, further enhancing its anti-bacterial capability.The preparation process of this material was shown in Scheme 1.
The Fourier Transform Infrared Spectroscopy (FTIR) spectrum of the PLLA@PDA/Cu composite fiber (Figure 1a) exhibited stretching vibration peaks of C = O at ≈1747 cm −1 , and C-O-C at 1076 cm −1 and 1179 cm −1 in PLLA. [24]The characteristic peaks at 1500 and 1275 cm −1 , respectively, indicated the N-H bending vibration and C-O stretching vibration peak of the phenolic moiety in PDA. [9]The weaker PDA phenolic peak appeared at 1275 cm −1 , suggesting that the reduction of Cu by PDA was completed. [22]From the X-ray diffractometer (XRD) analysis result of PLLA@PDA/Cu composite fiber (Figure 1b), it can be seen that the diffraction peak of Cu appeared in the composite fiber, which proved that PDA reduced Cu 2+ to Cu-NPs. [25]This was also proved by the Cu element distribution map (Figure 1c) that the distribution of Cu-NPs was relatively uniform, and the Cu element content was 2.38wt% (Figure 1d).As shown in the release profile of Cu 2+ of PLLA@PDA/Cu composite fiber (Figure 1e), a slight burst of Cu 2+ was observed in the first 1-3 d; with the prolongation of release time, the ion release tended to be stable, and reached 0.46 mg L −1 on the 10th day.Studies have shown that the effective concentration range of Cu 2+ to promote cell proliferation is 0.4 mg L −1 -4.0 mg L −1 .Therefore, the released Cu 2+ from PLLA@PDA/Cu composite fiber was within it, which can effectively promote cell proliferation.This is because the catechol group of PDA has a chelating effect on Cu 2+ , [25,27] achieving the slow and sustained-release of Cu 2+ .Therefore, the PLLA@PDA/Cu composite fiber was proved to hold excellent physiological stability.Pure PLLA fibers are more uniform in size and there was no adhesive phenomenon and no beads (Figure 2a).The diameter distribution was 80-160 nm, with an average diameter of 105 ± 5 nm (Figure 2b).The surface of PLLA@PDA composite fiber was partially covered by PDA film which relatively equally distributed (Figure 2c).For PLLA@PDA/Cu composite fiber, obvious spherical nanoparticles could be observed on the surface (Figure 2d), and distributed uniformly in a single fiber (Figure 1d) with a little small size (Figure 2e), The copper nanoparticle diameters were distributed in the range of 100-300 nm, with an average diameter of 183 ± 5 nm (Figure 2f).This may be attributed to the chelating effect between PDA and Cu 2+ .In this way, the "PDA-Cu 2+ " coordination bond was formed and Cu 2+ was further reduced to Cu-NPs due to the reducibility of PDA, [27,28] Figure 1.Characterization of composite fibers.a) FTIR spectra of PLLA@ PDA/Cu composite fibers; b) XRD maps of PLLA@PDA/Cu composite fiber; c) Elemental distribution of PLLA@PDA/Cu composite fiber; d) Elemental energy spectrum of PLLA@PDA/Cu composite fiber; e) Cu 2+ release profiles of PLLA@PDA/Cu composite fiber.making it more evenly distributed on the fiber surface.All these results demonstrated that the PLLA@PDA/Cu composite fiber was successfully engineered.

Hydrophilicity and Biological Activity of Composite Fibers
The surface hydrophilicity of the biomaterial is the main factor affecting cell adhesion and migration, and also affects the biological activity of itself [26] .As shown in (Figure 3a,b) (See Supporting Information) [20] , the water contact angles of PLLA, PLLA@PDA, and PLLA@PDA/Cu composite fibers were 116.1°, 54.4°, and 42.8°, respectively, which decreased in turn; The surface energy is 27.9, 49.02, 56.78 N m −1 , increasing in turn.Among them, the water contact angle of PLLA@PDA and PLLA@PDA/Cu fibers containing PDA was significantly smaller than that of PLLA fibers, which was due to the hydrophilic groups of hydroxyl and amino groups in PDA that helped to improve the hydrophilicity of composite fibers. [29]The superior hydrophilicity and strong adsorption capacity are beneficial to induce the deposition of bonelike apatite cells on the fiber surface, [9] and promote the biological activity of the fiber.Calcium (Ca) and phosphorus (P) are essential building blocks of bone.Elemental energy spectrum (EDS) analysis was applied to investigate the Ca and P elements when culturing osteoblasts on composite fibers for 10 d.As shown in Figure 3c-e, the PLLA@PDA/Cu composite fibers exhibited the highest contents of Ca and P with an amount of 0.83 and 3.88 wt.%, respectively.In contrast, there were only 0.07 wt% of Ca and 0.17 wt% of P in the PLLA fiber, and just increased to 0.43wt% and 0.72wt% in the PLLA@PDA group, respectively.This was attributed to the fact that the catechol group of PDA showed high ability to coordinate with Ca 2+ in the cell fluid, and could promote the formation of Ca and P on the surface of this material. [30]Meanwhile, Cu-NPs provided heterogeneous nucleation sites for calcium and phosphorus salts, increasing the mineralization of calcium and phosphorus salts.Therefore, PLLA@PDA/Cu composite fiber held the excellent biological activity.

Photothermal Properties of Composite Fibers
It has been reported that bacteria could be killed by safe 808 nm NIR photothermal therapy (PTT) in vitro/vivo without damaging normal tissues at ≈50 °C. [31]Under different power irradiation, the surface temperature of PLLA@PDA/Cu increased to 35.7, 48.2, 62.5, and 66.1 °C, respectively (Figure 4a), indicating that when irradiated for 10 min with 808 nm light at 1 W cm −2 , the NIR photothermal sterilization temperature can be reached, which was also within the acceptable temperature range of normal human tissues.This is because PDA and Cu-NPs, as PTT reagents, [32,33] absorbed the 808 nm NIR light, that is, the NIR light was absorbed by the PLLA@PDA/Cu fibers and then converted into heat through the photothermal effect.The surface temperatures of PLLA, PLLA@PDA, and PLLA@PDA/Cu composite fibers respectively reached 32.3, 39.1, and 48.2 °C after being irradiated at 1 W cm −2 (Figure 4b), demonstrating that the PLLA@PDA/Cu fiber can quickly approach the limit that the human body could bear under the 808 nm irradiation.
To measure and calculate the photothermal conversion efficiency of the composite fibers, the solid-state ultraviolet (UV) analysis was carried out from 200 to 1000 nm wavelength.As shown in Figure 4c, the absorbance of PLLA@PDA was 0.37, while that was 0.59 in the PLLA@PDA/Cu group at 808 nm.This may be due to the surface plasmon resonance (SPR) effect of Cu. [34] Besides, the photothermal conversion rate of PLLA fiber was 2.3% (Figure S3, Supporting Information) according to the temperature rise drop curve fitting; according to PLLA@PDA/Cu The photothermal conversion rate (See Supporting Information) [21] of Cu composite fiber was 23.7% (Figure 4d) by fitting the temperature rise drop curve, PLLA@PDA The photothermal conversion rate of the composite fiber is 11.4% (Figure 4e), which is mainly due to the surface plasmon resonance effect of Cu NPs, which can enhance the photothermal effect and photothermal conversion efficiency of the composite fiber. [35]In the heating-cooling cycle detection (Figure 4f), it was observed that the maximum temperature increased slightly in each cycle, which was caused by the volatilization of water in the system when the local temperature elevated, verifying that the PLLA@PDA/Cu showed excellent photothermal stability.
A large amount of ROS was generated on the surface of the fiber after NIR irradiation.And the production amount of singlet oxygen ( 1 O 2 ) was used to evaluate the content of ROS excited by NIR light.The results in Figure 4g exhibited that the absorbance of the PLLA@PDA/Cu at 410 nm decreased with the increasing irradiation time, indicating that the more 1 O 2 was produced, the more it was reacted with 1,3-diphenyliso -benzofuran (DPBF), the more the curve dropped.Interestingly, the absorbance of PLLA@PDA/Cu at 410 nm was significantly lower than that of PLLA and PLLA@PDA composite fibers (Figure 4h), since the SPR function of Cu-NPs produced electron-hole pairs, [36] which enabled to absorb H 2 O and O 2 , and further catalyze them to generate ROS with the highest yield.Therefore, it is demonstrated that PLLA@PDA/Cu composite fibers can effectively generate 1 O 2 .
Electron paramagnetic resonance spectroscopy (EPR) was used to test the generation of hydroxyl radical•(OH) and superoxide anion(O 2− ) on the surface of PLLA@PDA/Cu composite fibers.The PLLA@PDA/Cu composite fibers exhibited weak EPR signals without light.After 5 min of NIR irradiation at 808 nm, the EPR peak of•OH and•O 2− were observed.The EPR peaks of •OH (Figure 4i) and•O 2− (Figure 4j) were distinct, indicating the production of•OH and•O 2− .According to these results, the PLLA@PDA/Cu composite fibers can increase the production of ROS and produce various ROS under light, which can contribute to the antibacterial process.

Antibacterial Effect of Composite Fibers
After the bacteria were incubated on the fiber surface for 24 h, the qualitative analysis results shown in Figure 5a revealed that the bacteria on the surface of the PLLA fiber had vigorous metabolism and formed accumulated colonies, however, the  number of colonies decreased in the PLLA@PDA fibers, furthermore no colonies was found on the surface of PLLA@PDA/Cu fibers.For the quantitative results (Figure 5b), the antibacterial rates of PLLA, PLLA@PDA and PLLA@PDA/Cu fibers against E. coli were respectively 0, 64.3% and 100%, and reached 0, 97.1%, or 100% against S. aureus.The best antibacterial efficiency of PLLA@PDA/Cu benefited from the certain antibacterial properties of both PDA and Cu 2+ . [37]To verify the synergistic effect of light and heat on antibacterial, E. coli and Staphylococcus aureus (S. aureus) were incubated on the surface of the compos-ite fiber for 3 h.The qualitative results showed (Figure 5c) that when the PLLA@PDA/Cu fiber was not irradiated, the surface colonies grew vigorously, however, no colonies were observed on its surface after irradiation.The quantitative results in Figure 5d displayed that the inhibitory rates of PLLA@PDA/Cu composite fibers against E. coli and S. aureus were as high as 99% and 94%, respectively.The above results showed that the PLLA@PDA/Cu composite fiber exhibited synergistic antibacterial effect with PTT on E. coli and S. aureus, which greatly shortened the antibacterial time from 24 to 3 h, and potentiated the inhibitory rates to 99% and 94% after light irradiation compared to no irradiation group, achieving fast and powerful antibacterial efficacy.
Because Cu 2+ has an excellent antibacterial property, the electrostatic interaction between Cu 2+ and the negative charge on the surface of the bacterial membrane reduces the electrochemical potential and affects the integrity of the membrane. [38]Pits form in the bacterial membrane, causing the leakage of bacterial components from the bacteria's interior, which leads to oxidative stress and bacterial death. [39]Cu 2+ released from Cu-NPs could interact with thiol proteins, destroying bacterial and enzymatic proteins and disrupting the helical structure of DNA molecules. [40]At the same time, Cu-NPs had a photothermal effect, which could make water generate more ROS under the catalytic action of NIR light.ROS can cause oxidative damage to the membrane structure by attacking the bacterial membrane, resulting in the leakage of cellular contents.The ROS also led to the oxidation of proteins and the degradation of DNA, which killed bacteria. [41]Cu-NPs absorbed NIR light and converted to heat energy, utilizing the high temperature to inhibit the activity of bacteria and further accelerate the antibacterial activity.The chemical and photothermal actions of Cu-NPs cooperated to exert the antibacterial activity of this composite fiber.Subsequently, the Cu-NPs were freed from dead bacteria and continued to contact with other colonies.This repeated process helped the PLLA@PDA/Cu composite fiber to maintain its long-lasting antibacterial activity.And the mechanism diagram was shown in Figure 5e.

In Vitro Cellular Morphology and Activity of Composite Fibers
Osteoblasts are functional cells for bone formation, therefore the biocompatibility between fibers and osteoblasts needs to be investigated.As shown in Figure 6a, when cultured from 1 to 7 d, the osteoblasts spread completely on each different composite fiber, grew well, appeared pseudopodia and stretched around.The results of Cell Counting Kit-8 (CCK-8) cell viability assay displayed (Figure 6c) that there was no significant difference in the metabolic activity of osteoblasts in each group of composite fibers after 1 day incubation.When cultured for 3 d, no obvious difference in the cell activity of PLLA@PDA and PLLA@PDA/Cu composite fibers was observed.However, while cultured for 5-7 d, the metabolic activity of osteoblasts on PLLA@PDA/Cu was remarkably higher than that of PLLA@PDA and PLLA groups.In general, the metabolic activity of osteoblasts in each group of composite fibers increased over culturing time, and the cell activity on the PLLA@PDA/Cu fibers was significantly higher than that of other two fibers, which was more beneficial to osteoblasts and showed better biocompatibility to osteoblasts.This is because Cu 2+ released from PLLA@PDA/Cu fibers induced the generation of a hypoxic microenvironment.It has been reported that hypoxia could upregulate the expression of Annexin2-S100A10 complex in osteoblasts, which contained Ca 2+ binding sites, and the increment of internal Ca 2+ would promote the proliferation of osteoblasts. [10]Meanwhile, Cu 2+ also affected the expression of OCN, BMP-2 and other osteogenic-specific proteins, thereby potentiating osteogenesis.Furthermore, PDA improved the hydrophilicity of fibers, which was conducive to stimulating the proliferation and adhesion of osteoblasts. [42]The detailed mechanism was shown in Figure 6e.
Human bone contains a large number of microvessels for transporting nutrients, so the cytotoxicity of fibers to vascular endothelial cells (VECs) was also assessed in this study.It can be observed from Figure 6b that VECs adhered to the surfaces of PLLA, PLLA@PDA, and PLLA@PDA/Cu composite fibers and protruded pseudopodia from 1 to 7 d of culture.On the 7th day, VECs were completely covered the surface of PLLA@ PDA/Cu composite fibers cells.The CCK-8 detection data revealed (Figure 6d) that the cell activity on the PLLA fiber group was significantly much higher than that of the other two groups (PLLA@PDA and PLLA@PDA/Cu) from 1-3 d.However, when incubated for 5-7 d, the metabolic activity of VECs enhanced and showed the greatest activity in the PLLA@PDA/Cu group compared with the PLLA and PLLA@PDA composite fibers.Since the presence of the hydrophilic groups, including hydroxyl and amino groups in PDA, enhanced the growth and proliferation of VECs.PDA could also regulate the phosphorylation of vascular endothelial growth factor receptor-2 (VEGFR-2) during angiogenesis, thereby enhancing the adhesion and proliferation capacities of VECs. [43]Besides, the stable release of Cu 2+ further promoted the expression of HIF-1 and the secretion of VEGF from bone marrow stromal cells, thus stimulating the proliferation and differentiation of VECs. [10]Therefore, the PLLA@PDA/Cu composite fibers exhibited superior cytocompatibility and angiogenesis induction ability.

In Vitro Osteogenic and Angiogenesis Effect of Composite Fibers
To investigate the osteogenic and angiogenesis effect of PLLA@PDA/Cu composite fibers, western blot analysis was conducted to detect the related proteins, including BMP-2, OCN, S100A10, HIF-1, and VEGF.The results in Figure 7a-f showed that the protein expressions of cells treated with PLLA, PLLA@PDA, or PLLA@PDA/Cu exhibited an upregulation trend.Moreover, the protein expression of BMP-2, OCN, S100A10, HIF-1, and VEGF were the highest in the PLLA@PDA/Cu group.The results proved that Cu 2+ could effectively promote bone regeneration and angiogenesis in the process of bone defect repair.The addition of copper enhanced the ability of bone formation and angiogenesis.
Alkaline phosphatase was an early marker of osteogenic differentiation, ALP staining was used to detect the osteogenic properties of the fiber after being cultured with cells for 7 d.As shown in Figure 7g, the positive ALP staining of PLLA fiber, PLLA@PDA fiber and PLLA@PDA/Cu fiber increased gradually, and was the strongest in the PLLA@PDA/Cu group, indicating that PLLA@PDA/Cu fiber held the best bone-promoting ability.
Calcium salt deposition is one of the signs of bone formation in the late stage of osteogenesis.ARS was applied to detect calcium salt deposition of the fiber after 14-day incubation with cells.In Figure 7h, PLLA@PDA/Cu fiber displayed the strongest positive ARS intensity, compared to both PLLA fiber and PLLA@PDA fiber.This is because Alizarin red could specifically bind to the calcium nodules, [44] which were deposited on the surface of the  fiber, revealing that PLLA@PDA/Cu fiber had the most superior ability to accelerate bone regeneration.
Both ALP staining and ARS staining indicated that PLLA@PDA/Cu composite fibers had the best osteogenic effect in the early and late stages.

In Vivo Cytotoxicity of Composite Fibers
To study the biocompatibility of composite fibers, osteoblast extract was injected into a rat vein.After ten days, all mice were euthanized, and the myocardium, liver, lung, kidney, and skeletal muscle were processed for slicing, HE staining and histological analysis (Figure 8a-e).Results showed that the morphologies of these organs were ordered, and the structures were complete.Though some side effects were observed in the lung and liver, they were limited during the process of promoting bone regeneration in rats.The biocompatibility of PLLA@PDA/Cu was re-lated to the release behavior of Cu 2+ .PDA was used as a chelating agent of Cu 2+ to control the release rate of Cu 2+ .Therefore, PLLA@PDA/Cu composite fiber was biocompatible in this study.

Conclusion
Overall, in this study, the NIR-responsive PLLA@PDA/Cu composite fiber with rapid antibacterial and osteoinductive properties was engineered via a combination of electrospinning and self-assembly techniques.It was proved that the nanoparticles on the surface of the PLLA@PDA/Cu fiber were evenly distributed.And the biological activity study verified that hydrophilicity of this composite fiber was improved due to the interfacial concentration effect of PDA on Ca 2+ .And the Cu-NPs had a heterogeneous nucleation effect, and the fiber showed excellent bioactivity.Studies of the photothermal performance demonstrated that attributed to the responsiveness of PDA and Cu-NPs to 808 nm NIR light, the photothermal conversion rate of PLLA@PDA/Cu fiber reached 23.7%, suggesting its superior photothermal ability and stability.Furthermore, the antibacterial test results exhibited that the bacteria incubated on the PLLA@PDA/Cu composite fiber for 3 h, the antibacterial rates against E. coli and S. aureus were both 0 when not illuminated, however, respectively reached 99% and 94% after being irradiated, indicating that lighting greatly shortened the antibacterial time and achieved a rapid and powerful bactericidal efficacy.Osteoblasts and VECs culture showed that the PLLA@PDA/Cu fiber was favorable for cell adhesion and spreading, exhibited excellent cytocompatibility, and had good osteoinductive and angiogenesis abilities.Therefore, PLLA@PDA/Cu composite fiber is a promising photothermal antibacterial material in the treatment of bone defects.

Experimental Section
Detailed experimental materials and methods can be found in the Supporting Information.All animal experiments were performed in accordance with the guidelines for the management and use of laboratory animals in the First Affiliated Hospital of Xinjiang Medical University, and were approved by the Animal Ethics Committee of the First Affiliated Hospital of Xinjiang Medical University, Ethics Approval Number: IACUC-20170222054.

Figure 2 .
Figure 2. Characterization of composite fibers.a) Scanning electron microscopy (SEM) image of pure PLLA; b) Diameter distribution of PLLA; c) SEM image of PLLA@PDA; d) SEM image of PLLA@PDA/Cu; e) SEM enlarged image of PLLA@PDA/Cu; f) Copper nanoparticle diameter distribution in PLLA@PDA/Cu.

Figure 3 .
Figure 3. a) Water contact angle of composite fibers; b) surface energy of composite fibers; c-e) respectively: EDS-mapping element distribution and EDS energy spectrum of osteoblasts cultured on PLLA, PLLA@PDA and PLLA@PDA/Cu fibers for 10 d.

Figure 4 .
Figure 4. Photothermal performance analysis.a) The temperature rise curve of PLLA@PDA/Cu composite fiber after 10 min of 808 nm laser irradiation at different powers; b) The temperature rise curve of 808 nm laser irradiation for 10 min (1 W cm −2 ); c) The solid UV absorption spectrum of the composite fiber; d) The temperature rise and temperature fall curve of PLLA@PDA/Cu composite fiber and the fitting diagram of the cooling stage; e) The heating-up and cooling-down curves of PLLA@PDA composite fibers and the fitting diagram of the cooling-down phase; f) The cyclic heating-up curves of PLLA@PDA/Cu composite fibers (5 switching cycles); g) Detection data of 1 O 2 of fibers under different light times; h) Detection data of 1 O 2 at 10 min of light; i) ESR spectra of PLLA@PDA/Cu for the detecting•OH in the presence of DMPO; j) ESR spectra of PLLA@PDA/Cu for the detecting •O 2− in the presence of DMPO.

Figure 5 .
Figure 5. Plots of antimicrobial properties of composite fibers tested.a) General qualitative data plot; b) General quantitative data plot; c) Photothermal qualitative data plot; d) Photothermal quantitative data plot; e) Copper chemical signal and photothermal synergistic antibacterial mechanism plot.

Figure 6 .
Figure 6.a) SEM images of osteoblasts cultured in composite fibers for different days; b) SEM images of vascular endothelial cells cultured on the surface of composite fibers for 1, 3, 5, and 7 d; c) metabolic activity of osteoblasts on composite fibers; d) cellular activity of vascular endothelial cells on composite fibers.(* represents significant difference, P < 0.05); e) Osteogenic mechanism of PLLA@PDA/Cu composite fibers.

Figure 7 .
Figure 7. a-f) Detection of BMP2, OCN, S100A10, HIF-1 and VEGF protein expression by Western Blot (★Represents a significant difference, P < 0.05); g) ALP stained images of osteoblasts cultured on different composite fiber surfaces after 7 d; h) Alizarin stained images of osteoblasts cultured on different composite fiber surfaces after 14 d.