3D printed PCLA scaffold with nano‐hydroxyapatite coating doped green tea EGCG promotes bone growth and inhibits multidrug‐resistant bacteria colonization

Abstract Objectives 3D‐printing scaffold with specifically customized and biomimetic structures gained significant recent attention in tissue engineering for the regeneration of damaged bone tissues. However, constructed scaffolds that simultaneously promote bone regeneration and in situ inhibit bacterial proliferation remains a great challenge. This study aimed to design a bone repair scaffold with in situ antibacterial functions. Materials and Methods Herein, a general strategy is developed by using epigallocatechin‐3‐gallate (EGCG), a major green tea polyphenol, firmly anchored in the nano‐hydroxyapatite (HA) and coating the 3D printed polymerization of caprolactone and lactide (PCLA) scaffold. Then, we evaluated the stability, mechanical properties, water absorption, biocompatibility, and in vitro antibacterial and osteocyte inductive ability of the scaffolds. Results The coated scaffold exhibit excellent activity in simultaneously stimulating osteogenic differentiation and in situ resisting methicillin‐resistant Staphylococcus aureus colonization in a bone repair environment without antibiotics. Meanwhile, the prepared 3D scaffold has certain mechanical properties (39.3 ± 3.2 MPa), and the applied coating provides the scaffold with remarkable cell adhesion and osteogenic conductivity. Conclusion This study demonstrates that EGCG self‐assembled HA coating on PCLA surface could effectively enhance the scaffold's water absorption, osteogenic induction, and antibacterial properties in situ. It provides a new strategy to construct superior performance 3D printed scaffold to promote bone tissue regeneration and combat postoperative infection in situ.

Conclusion: This study demonstrates that EGCG self-assembled HA coating on PCLA surface could effectively enhance the scaffold's water absorption, osteogenic induction, and antibacterial properties in situ. It provides a new strategy to construct superior performance 3D printed scaffold to promote bone tissue regeneration and combat postoperative infection in situ.

| INTRODUCTION
3D bioprinting technique has been widely used to manufacture tissues and organoids, such as skin, bones and blood vessels, which had drawn much attention in regenerative medicine. [1][2][3][4][5][6][7][8] However, the application of 3D bioprinting scaffolds in tissue engineering has usually been hampered because scaffolds possess a smooth surface that limits cell adhesion, and its printing process usually requires the assistance of high temperature or organic solvent, making it difficult to integrate bioactive molecules on the scaffold during the printing. 9 To address this issue, many polymer or natural materials have been developed for fabricating scaffolds to increase their potential for biomedical applications. 10,11 High molecular polymer materials were considered as bone substitutes owing to their stiffness could meet the mechanical properties of the implantation site, helping to regenerate damaged areas. However, they had several inherent deficiencies when used as fillers in vivo, including low biological activity, poor osteoconductivity and inducibility, and possible immune responses as foreign objects. 12,13 Therefore, the design and construct a multifunctional 3D printed scaffold for bone repair and bone regeneration is urgently needed.
Currently, several methods are under study to endow bone repair scaffolds with bone regeneration properties, such as modifying the composition of biomaterials and using specific growth factors to stimulate cell adhesion and guide new tissue formation. 14,15 Among these approaches, some multiphase scaffolds with proper mechanical capabilities were manufactured for bone regeneration and bone repair. 16 However, few of these scaffolds could simultaneously achieve bone regeneration and in situ anti-infection. 17,18 It is reported that 10% of biomaterial-mediated bone repair was accompanied by bacterial infection after surgery, 19 such as osteomyelitis caused by Staphylococcus aureus. Not to mention the formation of bacterial biofilms on the scaffold surface, where colonizing bacteria is prone to develop resistance to antibiotics. 20 Clinically, patients are treated with antibiotics after bone repair surgery to prevent or treat bacterial infections at the site of the bone defect. However, the overuse of antibiotics will not only cause physical damage to patients, but also trigger an outbreak of multidrug resistant bacteria, posing a serious threat to human health worldwide. [21][22][23] Hence, a coating with dual biological activities of antibacterial and osteogenesis, combined with high-performance polymer to repair bone defects, is urgently desired.
An ideal strategy is to use natural bioactive molecules to integrate into scaffolds, giving them multifunctional properties. 24 Polymerization of caprolactone and lactide (PCLA) is a macromolecular copolymer prepared by bulk ring-opening polymerization of caprolactone (PC) and lactide (LA) monomers and has been exploited for medical application via controllable temperaturesensitive hydrogel or drug delivery capsules. 25,26 The degradation rate of the copolymer could be controlled by the monomer and reduces the acidity of the degradation products, which is an ideal scaffold for bone support and cell adhesion. The acicular nanohydroxyapatite (HA) can increase additives' biomineralization and cell proliferation ability. 27 If HA is integrated into the PCLA scaffold, it will confer multifunctional properties to the scaffold. We previously reported that the ethoxy group of the silane coupling agent was hydrolyzed to form a silanol reaction with the hydroxyl group of HA, resulting in a silicon-oxygen bond formation, and thereby enhancing the interface compatibility between HA and polymers, making it possible to modify HA onto PCLA. 28 As the first botanical prescription drug approved by the FDA, Veregen (PolyphenonE, a green tea extract) is used to treat genital warts caused by human papillomavirus infection. Epigallocatechin-3-gallate (EGCG) is the most biologically active and abundant polyphenolic compound in green tea, which possesses antibacterial, antiviral, anti-inflammatory, antitumor and immunemodulating activities. 29 Therefore, we hypothesize that using a silane coupling agent (KH550) as a linking agent to modify HA with EGCG and integrated it into PCLA may be applied for the regeneration of bone defects by relying on the advantages of EGCG and hydroxyapatite.
In this study, a PCLA scaffold was manufactured by using polyvinyl alcohol as the 3D printing to imitate the periosteum.
Then, a general method was developed by using EGCG selfassembled anchored in the HA with KH and coating the PCLA scaffolds (PCLA/KH-HA-EGCG). Taking advantage of the com-

| Characterization of the 3D porous scaffold
The surface morphology of the scaffolds was characterized using scanning electron microscopy (SEM, Phenom Pro, Netherlands).
The functional groups were identified by Fourier-transform infrared spectroscopy (FTIR; Thermo Fisher, Nicolet™ iS™ 10, Shanghai, China). Before performing FTIR, the nanoparticle sample must be vacuum dried first and then mixed with KBr powder to form a thin sheet for testing. The average particle sizes of EGCG modified HA were

| EGCG connection rate study
All the filtrate of initially prefabricated nano-particles was collected together. EGCG loading percentage in nano-particles was measured spectrophotometrically at 275 nm. The connection efficiency is calculated by the following equation: EGCG loading content %wt=wt ð Þ¼ mass of EGCG used À EGCG in sloution ð Þ Â 100 mass of EGCG used : where As: absorbance value of the scaffolds treatment group. Ac: Absorbance value of the control group without scaffolds. Ab: Absorbance value of the blank group without cells and scaffolds.

| RT-PCR
The pre-sterilized scaffold samples were placed into a 6-well plate with 2 ml/well of fresh MEMα medium to soak for 30 min. Then replace the medium used for infiltration with 2 ml of cell-containing MEMα medium.

| Hemolysis assay
The potential toxicity of the PCLA scaffolds to mouse red blood cells was assessed using a standard protocol. An animal experiment was conducted in compliance with the Chinese Academy of Medical Sciences guidelines and was approved by the Institutional Animal Care and Ethics Committee (Approval No. SCXK2014-0004). Briefly, a volume of 1 ml of blood collected from ICR mouse was put into the blood-collecting vessel. Then, the blood cells were collected using a centrifuge at 1500 rpm for 3 min. Next, samples were washed with PBS and diluted to 10 ml. After that, 1 ml diluted red blood cell suspension solution was mixed with PCLA, PCLA/ KH-HA, PCLA/HA-EGCG, and PCLA/KH-HA-EGCG scaffolds. These samples were then placed in a cell incubator at 37 C for 2 h. The positive control was added deionized water. Next, these scaffolds were removed from the samples before centrifugation. Finally, samples were photographed and the absorbance of supernatant at 570 nm was measured using a microplate reader to obtain the hemolysis percentage. The relative hemolysis percentage was calculated by the following equation:
All data quantifications were done on high-resolution images using Image Pro-Plus6.0 software.  Figure 2F). Additionally, particle sizes of KH-HA, HA-EGCG, and KH-HA-EGCG fabricated were also investigated. The particle sizes decreased as the EGCG content increased ( Figure 2D-F). This may be attributed to the HA nanoparticle being firstly treated with the silane coupling agent to enhance their dispersibility, and then the amino group inside the silane coupling agent was bridging the EGCG to the HA surface. These results collectively proved that EGCG was successfully modified on the surface of HA, and KH-HA-EGCG had a higher EGCG intensity.

| Fabrication and characterization of 3D coated scaffold
The open pores in the scaffold directly affect the transport of nutrients and metabolites for bone tissue growth, as well as the formation of new blood vessels. [30][31][32] Our previous research reported that the prepared PTMC/KHA/VH microsphere scaffold had 42% porosity and 100 μm pore size using microsphere sintering technology. 28 This PTMC/KHA/VH microsphere scaffold presented bone repair performance with good biocompatibility in vitro and in vivo. However, the PTMC/KHA/VH scaffold had poor repeatability due to the microspheres' wide particle size distribution range of the microspheres. The application of 3D printing technology can accurately customize any geometrical bone repair for the patient's injury site, and can also effectively control and optimize the porous structure of the scaffold to optimize bone healing. 33 In this study, we fabricated the scaffolds using a 3D printer with a nozzle diameter of 200 μm. The PCLA scaffold was firstly manufactured in a prefabricated mesh model and used 0/90 or 0/45 mode to continuously extrude polymer filaments under a pressure of 200 kpa to ensure the 3D porous space structure of the printed scaffold. This process was used as a single-layer printing of the scaffold (Video S1). After many times layer-by-layer printing and assembly, a 3D scaffold with a height of millimetres or centimetres can be quickly printed. 34 During the coating process, ( Figure 3B). On the contrary, it is obvious that the surface of the coated scaffold is very rough, and the presence of HA could be observed. Interstingly, PCLA/KH-HA-EGCG scaffold exhibits a more uniformly dispersed microstructure ( Figure 3C-E). This is because the smaller the particle size of KH-HAZ-EGCG, the better its dispersion in the solution at the same concentration.
Based on the different appearances of the scaffold coating, we further quantified the coating content of the 3D printed PCLA scaffold surface. The 3D scaffold sample matched the coating very well during the coating process. As depicted in Figure 4A, calcium ion content was quantified to evaluate the coating efficiency of modified PCLA scaffolds. PCLA/HA-EGCG and PCLA/KH-HA-EGCG groups presented higher concentrations of calcium ions than PCLA/KH-HA ( Figure 4A). This was because the particle size of HA-EGCG and KH-HA-EGCG were smaller ( Figure 2D-F), and the amount of HA adhered to the PCLA scaffolds was higher at the same concentration. EGCGmodified HA coating designed in this study could stably exist on the surface of the scaffold to achieve long-term effects. As excepted, the quantitative results of the coating showed that the coating could stably exist on the surface of the scaffold, whether in PBS buffer, MEMα medium, or absolute ethanol and could maintain stability for a long time ( Figures 4B,C, S4, and S5). The Young's modulus of PCLA scaffold was 39.3 ± 3.2 MPa, which has similar mechanical properties to cartilage (20-100 MPa; Figure 4D). 35 Notably, the coating method designed in this study could be useful for any 3D printed scaffold with different applications. Furthermore, the 3D scaffold samples' porosity was measured using absolute ethanol replacement technology. The porosities of PCLA, PCLA/KH-HA, PCLA/HA-EGCG and PCLA/KH-HA-EGCG scaffolds were 60.7 ± 0.6%, 60.4 ± 1.7%, 60.8 ± 0.6%, and 61.6 ± 0.9%, respectively ( Figure 4D). There was no significant difference in pore connectivity and porosity between the scaffolds with different coatings. The water uptake of scaffolds fabricated with various coating was also investigated. The application of the coating for the surface of the scaffold fabricate had enabled increased scaffold's water absorption performance to optimize buffer infiltration and facilitate cell adhesion. As shown in Figure 4D, the water absorption of the 3D scaffold increased when the coating was KH-HA-EGCG nanoparticles. This is because the rough structure makes the PCLA scaffold surface have a larger specific surface area, making it difficult to remove excess water. Moreover, HA has a strong ability to absorb water and release water and absorb moisture in the air. 36 This increase in water absorption is different from our previous studies on the water absorption of the bone repair scaffold with the incorporation of modified inorganic particles. 28 On the other hand, we evaluated the cytotoxicity of these coated scaffolds. As depicted in Figure 4E, after treatment with different coating PCLA scaffolds, the count of HepG2 cells did not decrease, indicating that these scaffolds had good biosafety for HepG2 cells.
Cell motility is an important indicator for evaluating cell viability. 37,38 Wound-healing studies showed that the motility of HepG2 cells was not affected by different PCLA scaffolds treatment, further proving that these scaffolds were not toxic to cells ( Figure 4F,G). As expected, coated scaffolds did not exhibit cytotoxicity to A549 cells ( Figure S6).
Furthermore, we used a hemolysis experiment to evaluate the toxicity of scaffolds materials on the rupture and lysis of red blood cells in ICR mouse. PCLA/KH-HA-EGCG did not cause obvious hemolysis, indicating their excellent blood biocompatibility ( Figure 4H).

| PCLA/KH-HA-EGCG scaffold inhibits bacteria in vitro
Staphylococcus aureus (SA) is one of the main causes of osteomyelitis. Moreover, multiple drug-resistant bacteria MRSA were often detected clinically, posing a threat to human health. [39][40][41] In this study, the zone of inhibition (ZOI) study was performed to assess the anti-MRSA activity of prepared materials. 42 As depicted in To investigate the antibacterial mechanism of the coated scaffolds, the morphology and structural integrity of the treated MRSA in different groups were observed by SEM. As depicted in Figure 5B, the wall and membrane structure of MRSA treated with PCLA/KH-HA-EGCG had been seriously destroyed. When the structure of bacteria is damaged, its intracellular components will flow out of the cell, such as nucleic acid and proteins. As shown in Figure 5D, Excessive ROS will attack important macromolecules in bacterial cell (e.g., nucleic acid, proteins and lipid), and eventually cause cell death. 43,44 As depicted in Figure 5E, with the blank control group, the number of MC3T3-E1 cells stimulated by KH-HA, HA-EGCG, and KH-HA-EGCG nanoparticles was increased at the same coating content concentration ( Figure 6A). This is mainly because the Ca 2+ and PO 4 3À ions released by HA are beneficial to stimulate the proliferation of bone cells. Moreover, HAmodified PCLA scaffolds also had the ability to promote MC3T3-E1 osteoblast cells proliferation. As shown in Figure 6B, MC3T3-E1 cells showed significant proliferation after PCLA/KH-HA, PCLA/HA-EGCG, and PCLA/KH-HA-EGCG scaffolds treatment. We also performed a live cell ratio test to investigate the detailed cell survival rate on the scaffolds, which showed PCLA/KH-HA-EGCG scaffold presented an excellent proliferation state ( Figure 6C). To further intuitively reveal the effect of scaffolds on MC3T3-E1 cell proliferation activity, we cultured mouse MCT-E1 cells with different coated scaffolds and imaged them with a fluorescence microscope. After culturing for 1, 3, 5 ( Figure S7) and 10 days ( Figure 6D), live/dead staining showed that the cells seeded on the coated scaffold had more proliferation (green), and few dead cells (red) were observed. In this study, HA is the main component of human bone tissue, and it is also the main bio-inorganic particle used in the current research on promoting bone regeneration. 45 When it is compounded on the surface of PCLA scaffold as a coating, calcium and phosphorus will be freed from the surface of the coated scaffolds. The calcium and phosphorus will be absorbed by adherent cells, stimulating cell differentiation and even new tissue formation. Together, we demonstrate that after adding EGCG-modified HA coating on the surface of the scaffold, the 3D printed PCLA scaffold could effectively promote cell adhesion and proliferation.
Furthermore, we analysed the biomineralization efficiency of the scaffold by observing the formation of the hydroxyapatite layer on the surface of the scaffold in a SBF environment, which was an essential feature of an ideal bone repair scaffold. 46   The ALP activity of mouse MC3T3-E1 was examined on days 3 and 7 ( Figure 7D). According to the normalized quantitative data analysis, cells seeded in the PCLA group showed significantly lower ALP activity after the cell culture period. The ALP activity of all scaffold groups was the same on the first day and gradually increased with the pro-

| CONCLUSIONS
We demonstrated that manufacturing a 3D structural scaffold using an extrusion-based printing method and imparting an EGCG-modified HA coating on its surface could effectively enhance the scaffold's water absorption, osteogenic induction and antibacterial properties in situ. In this study, simple HA mixture solutions were used to disperse