Zinc Doped Halloysite Cooperating with Polylactic Acid for Bone Regeneration

Three-dimensional (3D) printing techniques have received considerable focus in the area of bone engineering due to its precise control in the fabrication of complex structures with customizable shapes, internal and external architectures, mechanical strength, and bioactivity. In this study, we design a new composition biomaterial consisting of polylactic acid (PLA), and halloysite nanotubes (HNTs) loaded with zinc nanoparticles (PLA+H+Zn). The hydrophobic surface of the 3D printed scaffold was coated with two layers of fetal bovine serum (FBS) on the sides and one layer of NaOH in the middle. Additionally, a layer of gentamicin was coated on the outermost layer against bacterial infection. Scaffolds were cultured in standard cell culture medium without the addition of osteogenic medium. This surface modification strategy improved material hydrophilicity and enhanced cell adhesion. Pre-osteoblasts cultured on these scaffolds differentiated into osteoblasts and proceeded to produce a type I collagen matrix and subsequent calcium deposition. 3D printed scaffolds formed from this composition possessed high mechanical strength and showed an osteoinductive potential. Furthermore, the external coating of antibiotics not only preserved the previous osteogenic properties of the 3D scaffold but also significantly reduced bacterial growth. Our surface modification model enabled the fabrication of a material surface that was hydrophilic and antibacterial, simultaneously, with an osteogenic property. The designed PLA+H+Zn may be a viable candidate for the fabrication of customized bone implants.


Introduction
According to the reports of the National Ambulatory Medical Care Survey and American Academy of Orthopedic Surgeons, about 6.8 million patients ask for medical therapy due do orthopedic problems every year, and more than two million bone grafting procedures are performed annually. 1 Autografts are considered the gold standard for bone repair because of their excellent properties in osteoconduction, osteoinduction, and osteogenesis; 2 however, the concerns of limited supply, the required surgery at the site where the bone was harvested, and the risks of accompanied hematoma, infection, and additional pain from donor site afflict patients. 3 Allografts are another source for orthopedic implants, and nearly one-third of all bone grafts used in North America are allografts. 4 However, allografts are osteoconductive but with reduced osteoinductivity, which increases the risk of nonunion in fracture repair, and there is a risk of infection. 5,6 Also, the supply of allografts is limited by the long pretreatment process. Every year there remains a long list of patients waiting for receiving a bone allograft. A new method for fabricating a bone graft with proper mechanical properties, osteoconductivity, and inductivity could enhance osteogenesis is urgently needed.
Bone is a porous tissue with numerous interconnected pores that permit cell migration and proliferation, as well as vascularization; 7 therefore, an osteogenic scaffold should mimic bone morphology, structure, and function in order to ensure its integration with the native tissue.
Bone implants can be produced through a variety of methods: salt leaching 8 , chemical/gas foaming9, freeze-drying, 17 and sintering. 10 However, pore size, pore distribution, porosity, and pore interconnectivity cannot be precisely controlled with these approaches. 11 Threedimensional (3D) printing technology has received considerable attention for tissue regeneration due to its superiority in the fabrication of complicated structures with tailored shapes, internal and external architecture, pre-designed microstructure, mechanical strength, and bioactivity, which can effectively mimic native tissues. With the use of osteogenic biomaterials and computer-aided design, 3D printing technology can generate a customized structure with desired features that can improve bone integration and the restoration of tissue function. 7 Hybrid materials with tunable properties have been explored in 3D printing [12][13][14] . Polylactic acid (PLA) is a popular material used for 3D printing medical devices. It is a thermoplastic polymer that is derived from fermented corn starch, cassava starch, or sugarcane 15 . It is an eco-friendly bioplastic as it is entirely biodegradable and consists of renewable raw materials.
This material exhibits high tensile strength, low elongation, and high modulus, which enables it to be a suitable candidate for load-bearing applications, such as orthopedic fixation and sutures 15 .
In this study, PLA was used to fabricate porous scaffolds through 3D printing. According to previous reports, large pore size and high porosity are key factors in producing an osteogenic response. [16][17][18] Therefore, we designed the scaffold with an average pore size of 600 m and 60% porosity. PLA is a versatile, biodegradable, and FDA approved biomaterial 15 , but its surface is hydrophobic and, therefore significantly reduces cell adhesion. Accordingly, surface modification to enhance its cell supportive material properties can be achieved through the addition of micro-and nanoparticles 12 , fibers, 14 or fabrication of nanocomposites 13 . Halloysite is an aluminosilicate clay material Al2O3•2SiO2•nH2O; it forms nanoscale tubes during the formation process. Those nanotubes are in the length of 0.5-2 μm, and with a hollow lumen that is 10-15nm in diameter, the outer diameter range between 50-80nm 19 . Halloysite nanotubes (HNTs) are cyto-and biocompatible 20 . They have attracted increasing attention in biomedical research due to its physicochemical stability, the potential for chemically modification, and ease of doping substances within its lumen, including therapeutic agents 21-24 , enzymes 25 , nucleic acid 26 , and metal nanoparticles 27 . In addition, HNTs have been proven to enhance mechanical properties for numerous materials, such as alginate 25 , chitosan 28,29 , epoxy 30 , nylon 24 , rubber 31 , and calcium phosphate 32 . Furthermore, HNTs have also been reported to chemotactically attract pre-osteoblasts 33 and enhance osteogenic differentiation 34,35 .
Here, we used halloysite due to its known ability to improve polymer material properties and release bioactive agents in a sustained manner. HNTs were loaded with zinc nanoparticles.
Zinc is one of the essential minerals that play an essential role in bone health. It affects multiple enzyme activities, collagen synthesis 36 , and DNA synthesis 37 , and it has been demonstrated to stimulate bone metabolism 38 . Then, the zinc-loaded HNTs mixed with PLA for 3D printing. Fetal Bovine Serum (FBS) and NaOH were used to improve the surface hydrophilicity of a3D printed scaffold. Scaffold mechanical properties and cell-material interactions were studied. We also coated the 3D printed scaffold with antibody, gentamicin, to prevent contamination and assessed the drug efficiency after three weeks. This study aims to generate a 3D printed scaffold to support bone regeneration and prevent bacterial contamination, which may be potentially used for bone defect therapy in the clinic.

Zinc Loaded into HNTs
Zinc nanoparticles (NPs) were deposited on the HNT surface by thermal decomposition of the metal acetate, as depicted in Figure 1. Zinc oxide (ZnO) reacted with acetic acid at 50°C with continuous stirring, then the mixture was heated to a boil, and the reaction continued for 12 hours, with additional acetic acid added during this time period. The resulting zinc acetate (Zn(OAc)2) was filtered using Whatman #1 filter paper 39 . Then, 20g of Zn(OAc)2 mixed with 10g of HNTs in 50 ml DI water and stirred for 12 hours. After centrifugation, the pellet was collected and heated at 350°C for 2 hours, which led to thermal decomposition of the metal acetate on HNTs surface (ZnO-HNTs) 40 .

Material Preparation
Four composition were tested in this study: PLA, PLA+HNTs, PLA+HNTs/Zn, and PLA+HNTs/Zn+gentamicin. These groups were printed using an ENDER 3 printer with similar setting; however, different filament compositions were used. Filaments were extruded using a Noztek Pro Extruder (West Sussex, England) with a uniform diameter 1.750.05mm, but there was slightly different in filaments preparation for each group. For PLA group, PLA filaments were extruded at 175°C . For PLA+HNTs group, in order to archive a uniform distribution of HNTs in PLA, 10 l of silicon oil was added into 20g PLA and vortexed for 10 minutes, then 1.2g of HNTs were added and continually vortexed for another 10 minutes. Then mixture of PLA+HNTs were extruded at 170°C. Filaments of PLA+HNTs/Zn prepared similarly as PLA+HNTs; the only difference is HNTs were loaded with Zn (30% w/w) and extruded at 165 °C.
PLA+HNTs/Zn+gentamicin scaffolds were printed with PLA+HNTs/Zn filaments, and then they dipped into a 100mg/ml gentamicin solution for 24 hours.

3D Printing
All filaments types were 3D printed into a pre-designed structure (squares) using an ENDER 3 printer at 225°C. The squares were designed to be 662 mm with a pore size of 0.6mm ( Figure 2). The diameter of inside lattice supports was 0.6mm.

Porosity
The porosity of the 3D printed disks was calculated through liquid displacement. One 3D square was immersed into 1.0 ml (V1) of DI water, then a series of vortexing and sonication was applied to force the liquid into the pores. The total volume of square and DI water was measured (V2), after the water was removed, the square and the remaining volume of DI water was measured (V3). The final porosity of the square was calculated as below:

Compression Testing
A Univert CellScale Testing device (Waterloo, Ontario, Canada) was used for compression test of the printed squares. 3D printed squares were compressed at a speed of 10 mm/min with a 200 N load cell. The strain and stress profiles were recorded. A minimum of 3 tests were performed for each composition.

Surface Treatment of 3D Printed Square
According to the pilot study (Supplementary information), a sandwich coating ( Figure 3) on the 3D printed squares was shown to significantly improve the surface hydrophilicity and facilitate cell adhesion. Therefore, we coated the 3D printed disk for three layers. For the first layer, each square was immerged in fetal bovine serum (FBS) for 24 hours; then each square was immerged into 10 N NaOH for 30 minutes and washed three times with sterilized DI water; for the last layer, squares were incubated in FBS again for 24 hours. Squares with a three-layer sandwich-like coating were labeled as FBS+NaOH+FBS. Figure 3. The process of applying the three-layered coating onto 3D printed squares.

Morphology and Surface Characterization
The morphology of 3D printed disks was observed using a scanning electron microscope (SEM) and laser confocal microscope. The distribution of HNTs and HNTs/Zn in the PLA filament was observed by Energy Dispersive Spectrometer (EDS) using a PLA filament in cross-section.
The presence and nature of the surface coating was also determined by EDS.

Cell Proliferation
Surface modified squares were put into 48 wells plate, each well had one square and seeded with pre-osteoblast (MC3T3-E1, ATCC, Manassas, VA) at a cell density of 110 5 /well.

Picrosirius Red Staining
Picrosirius Red is a specific collagen fiber stain that is capable of detecting thin collagenous fibers. The media was aspirated from the cell culture plates, and each culture well was washed with DPBS before being fixed in 4% paraformaldehyde. Fixed cells were stained with Picrosirius Red to quantify the amount of collagen secreted. Picrosirius stain was added to each well and removed after an hour incubation at room temperature. The cells were rinsed with 0.5% acetic acid solution twice and absolute alcohol twice. Digital images of stained squares were acquired using a brightfield microscope. Cells cultured in monolayer were used as control.

Antibacterial Efficiency
The antibacterial ability of PLA+HNTs/Zn+gentamicin squares against Staphylococcus aureus (S.aureus) was assessed. 3D printed squares were placed in 24-well cell culture plate and each well was inoculated with 1ml S.aureus (0.3 x 10 7 CFU/ml). The S.aureus was sub-cultured from a single colony and maintained in Muller Hinton broth. The plate was incubated in horizontal orbital microplate shaker at 37°C for 12 hours. The absorbance of the incubation solution at 630nm was measured. Muller Hinton broth without PLA+HNTs/Zn+gentamicin squares and S.aureus set as negative and positive control, respectively.

Statistical Analysis
A one-way ANOVA or Student T-test was used for statistical analysis. Data were expressed as mean  standard error. A p-value less than 0.05 was considered statistically significant.

Distribution of HNTs and Zinc Nanoparticles in the PLA Filament
Filaments used to print PLA+HNTs, and PLA+HNTs/Zn squares were prepared by mixing PLA with HNTs or zinc-coated HNTs (HNTs/Zn). In order to determine whether the HNTs or HNTs/Zn were distributed throughout the PLA, filament cross-sections were analyzed with EDS. In Figure   4, all pictures represent the same visual field but present different elements. The primary element of PLA is carbon (C), which is exhibited all over the screen. Silicon (Si) and aluminum (Al) are the two major elements of HNTs, according to the graph, they were well distributed in the PLA filament. Zinc nanoparticles were coated into HNTs with 30% w/w, its distribution was detected by EDS as well. According to the EDS analysis, HNTs and HNTs/Zn were well distributed throughout the PLA filament.

Morphology of 3D printed Squares and their Surface Characteristics
All filaments were printed into a pre-designed square with a pore size of 600 m x 600 m and a layer height of 600 m (Figure 2). Due to the limitations of the 3D printer used, the resolution changed slightly during printing. The exact pore size was determined using a laser confocal microscope ( Figure 5). Based on the measurement of 60 pores from 20 different scaffolds, the average pore size of printed scaffolds is 584.1695.28 m x 620.3993.03 m and with a porosity of 60.229.5%.

Chemical Deposition
After processing the sandwich-like layered surface modification, the hydrophilicity of the printed squares was significantly improved (Supplementary Figure 1). In our hypothesis, surface hydrophilicity would keep increasing with each layer modification. However, the hydrophilicity decreased after the second layer modification was treated with NaOH, and then the hydrophilicity significantly increased after the third layer was added (Supplementary data, Figure 1). This phenomenon may have occurred because the NaOH eroded the chemicals that were deposited in the first layer modified with FBS. However, simultaneously, this erosion produced more links for chemical deposition, which lead to increased chemical deposition after the addition of the third layer (supplementary Figure 2, 3, and Figure 7).

Antibacterial Studies
FBS contains many substances which may lead to the growth of undesired microorganism; therefore, we coated the PLA+H+Zn with gentamicin (PLA+H+Zn+G). Gentamicin is an efficient antibiotic against gram-positive and negative bacteria41. Even though they were stored at 37°C for three weeks, they still efficiently inhibited bacterial growth ( Figure 8). As expected, printed squares without gentamicin showed no bacterial growth inhibition.

Response of Pre-Osteoblast to 3D Printed Squares Cell Proliferation
Many studies have shown that surface features, such as charge 42,43 , roughness 44 , adsorbed proteins, and hydrophilicity/hydrophobicity 45 , greatly influenced cell attachment and subsequent cell behaviors. Our results consist with previous studies, cell adhesion was improved with hydrophilicity and increased protein attachment (supplementary data, Figure 4).
In addition, cells preferred to proliferate on 3D squares as compared to than monolayer cultures ( Figure 9).
The influence of gentamicin on cell proliferation was also assessed. Consistent with the study of Philip et.al 46 , the presence of gentamicin did induce a small but transient effect on cell proliferation ( Figure 9). However, the presence of gentamicin did not have a negative effect on mineralization ( Figure 10).

Bone formation
Bone consists of bone cells and a mineralized collagenous matrix 47,48 . The main constituents of the bone matrix are hydroxyapatite [Ca10(PO4)6(OH)2] (50-70%) and an organic matrix (20-40%). 49 Type I collagen is the major component of bone tissue extracellular matrix (ECM), which is mainly synthesized by osteoblasts. The synthesis of type I collagen is one of the markers of osteogenic differentiation 50 . Processed with Picrosirius Red staining, type III collagen stained red, and type I collagen stained yellow. In the first 7 days incubation, collagen secretion by cells in monolayer culture was negligible. In contrast, type III collagen synthesized by cells cultured on different 3D square compositions was very apparent (Supplementary information Figure 5).
Furthermore, compared to the inner space, more type III collagen was synthesized on the bottom surface of the square, and the transformation from type III collagen to type I collagen happened earlier on the bottom surface of the well (Supplementary data Figure 5 vs. Figure 6).
Osteoblast differentiation in 3D scaffolds usually spreads from the scaffold periphery and gradually proceeds into the inner scaffold space 51 . Similar to the results seen with collagen synthesis, after 21 days of incubation, there was increased calcium deposition in the 3D scaffolds as compared to cells in monolayer culture ( Figure 10). Calcium deposition indicates mineralization of the bone matrix, which is another marker for bone tissue formation. Alizarin Red S stained the calcium deposited in the collagenous matrix (red), which was rarely found in monolayer culture after 7 days of incubation (Supplementary data Figure 7).

Conclusion
In this study, we 3D printed squares composed of PLA and zinc doped HNTs for use as a potential bone implant. The material's porosity mimicked that of human bone tissue. When a