Three-dimensional (3D) printing technology has received considerable attention in the field of bone engineering because it can precisely control the manufacture of complex structures with customizable shapes, internal and external structures, mechanical strength, and biological activity. In this study, the researchers designed a new composite biomaterial composed of polylactic acid (PLA) and kaolin nanotubes (HNTs) containing zinc nanoparticles (PLA+H+Zn).
On the surface of the hydrophobic 3D printed stent, two layers of fetal bovine serum (FBS) were coated on both sides, and a layer of sodium hydroxide was coated in the middle. Apply a layer of gentamicin on the outermost layer to prevent bacterial infection. The scaffold is cultured in standard cell culture medium, without the addition of osteogenic culture medium. This surface modification strategy improves the hydrophilicity of the material and enhances the adhesion of cells. The pre-osteoblasts cultured on these scaffolds differentiate into osteoblasts, which in turn produce type I collagen matrix and subsequent calcium deposition.
The 3D printed scaffold formed by the composition has high mechanical strength and has the potential to induce bone formation. In addition, the outer coating of antibiotics not only retains the bone-forming properties of the 3D scaffold, but also significantly reduces the growth of bacteria. The researcher’s surface modification model makes the preparation of the material surface hydrophilic and antibacterial, as well as osteogenic.
According to the National Mobile Health Care Survey and the report of the American Academy of Orthopaedic Surgeons, approximately 6.8 million patients request medical treatment for orthopedic problems each year, and more than 2 million bone transplants are performed each year. Autologous bone grafts are considered the gold standard for bone repair due to their excellent performance in bone conduction, osteoinduction and bone formation. However, autotransplantation has many limitations. These include limited availability, the need for surgical incisions to obtain the graft, which brings additional risks of hematoma, infection, and additional pain. Allogeneic bone grafts are another source of orthopedic transplantation. Nearly one-third of all bone grafts used in North America are allogeneic bone grafts. However, allogeneic bone has osteoconductive effect, but its osteoinductive ability is reduced, which increases the risk of fracture repair nonunion, and there is a risk of infection. In addition, the supply of allografts is also limited by the long pretreatment process. Based on the above reasons, researchers urgently need a new method of bioengineered bone graft with good mechanical properties, bone conductivity and osteogenic ability.
Bone implants can be produced by a variety of methods, including salt immersion, chemical/gas foaming, freeze drying, and sintering. However, these methods cannot precisely control pore size, pore distribution, porosity, and connectivity between pores. Bone is a porous tissue with many interconnected pores that allow cell migration and proliferation, as well as vascularization. Therefore, the osteogenic scaffold should mimic the shape, structure and function of bone to ensure its fusion with natural tissues. Three-dimensional printing technology has received extensive attention in the field of tissue regeneration because it can produce complex structures with customized shapes, internal and external structures, pre-designed microstructures, mechanical strength and biological activity, and can effectively simulate natural tissues. By using osteogenic biomaterials and computer-aided design, 3D printing technology can generate customized structures with desired characteristics, thereby improving osseointegration and tissue function recovery.
Here, the researchers used halloysite because of its known ability to improve the properties of polymer materials and continuously release bioactive agents. HNT is loaded with zinc nanoparticles. Zinc is one of the essential minerals that plays an important role in bone health. It affects the activity of a variety of enzymes, collagen synthesis and DNA synthesis, and has been shown to stimulate bone metabolism. Zinc oxide nanoparticles also have known and effective agents, which disintegrate bacterial cell membranes and accumulate in the cytoplasm leading to apoptotic cell death. Therefore, zinc was chosen as the coating of HNT and then mixed with PLA for 3D printing. Fetal bovine serum (FBS) and NaOH are used to improve the surface hydrophilicity of 3D printed stents. The mechanical properties and cell-material interaction of the scaffold were studied. The researchers also coated the 3D printed stent with the antibiotic gentamicin to prevent contamination, and evaluated the efficacy of the drug after three weeks. This research aims to produce 3D printed scaffolds to support bone regeneration and prevent bacterial contamination, which may be used in clinical treatment of bone defects.
HNTs
Nano-zinc (NPs) are deposited on the surface of HNT through the thermal decomposition of metal acetate, as shown in Figure 1. Zinc oxide (ZnO) reacts with acetic acid at 50° C., stirring is continued, and then the mixture is heated to boiling, during which time acetic acid is added, and the reaction lasts for 12 h. The obtained zinc acetate (Zn (OAc) 2) was filtered using Whatman #1 filter paper. Then, 20 g of zinc (OAc) 2 and 10 g of HNTs in 50 ml of deionized water were stirred for 12 h. After centrifugation, the particles were collected and heated at 350°C for 2 h, resulting in the thermal decomposition of metal acetate on the surface of HNTs (ZnO-HNTs).
3D printing
Use ENDER 3 printer to 3D print all filament types into a pre-designed structure (square) at a temperature of 225°C. The square design is 6×6×2 mm, the aperture is 0.6 mm, and the diameter of the inner grid is 0.6 mm.
Porosity
The porosity of the three-dimensional printed disc was calculated by the liquid displacement method. A 3D square is immersed in 1.0 mL (V1) of deionized water, and then the liquid enters the pores through a series of vortex and sound waves. Measure the total volume of the square sum DI water (V2), after removing the water, measure the remaining volume of the square sum DI water (V3).
Compression test
A single-scale unit test device in Waterloo, Ontario, Canada was used to perform compression testing on the printed grid. A 200n load cell was used to compress the 3D printed square at a speed of 10mm/min. The strain and stress distribution graphs are recorded. Test each ingredient at least three times.
3D printed square surface treatment
According to previous research (supplementary information), applying a layer of interlayer coating on a 3D printed square can significantly improve surface hydrophilicity and promote cell adhesion. Therefore, the researchers applied a three-layer coating on the 3D printed disk. Before coating, each disk was soaked in 75% isopropanol for 10 minutes and air-dried in a cell culture hood. In the first layer, each square was soaked in fetal bovine serum (FBS) for 24 hours; each square was soaked in 10n NaOH for 30 minutes and washed 3 times with sterile deionized water; in the last layer, the squares were incubated in fetal bovine serum for 24 hours again, The square with three layers of sandwich coating is marked as FBS+NaOH+FBS.
Distribution of HNTs and Zinc Nanoparticles in Polylactide Yarn
Mix PLA with HNTs or galvanized HNTs (HNTs/Zn) to make filaments for printing PLA+HNTs and PLA+HNTs/Zn squares. In order to determine whether HNTs or HNTs/Zn are distributed throughout the PLA, the filament cross-section was analyzed by EDS. The main element of polylactic acid is carbon (C), which is displayed on the screen. Silicon (Si) and aluminum (Al) are the two main elements of HNTs. It can be seen from the figure that they are well distributed in the PLA filament. The nano-zinc was coated in HNTs at 30% w/w, and its distribution was detected by EDS. EDS analysis showed that HNTs and HNTs/Zn were evenly distributed.
The shape and surface characteristics of 3D printed blocks
All the filaments are printed into a pre-designed square, with an aperture of 600 m×600 m, and a layer height of 600 m. Due to the limitation of the 3D printer used, the resolution changes slightly when printing. A laser confocal microscope was used to determine the exact aperture. By measuring 60 holes of 20 different scaffolds, the average pore diameter of the printed scaffold is 584.16±95.28 (m)×620.39±93.03 (m), and the porosity is 60.22±9.5%.
Compressive strength
In order to evaluate the contribution of HNTs to enhancing the mechanical properties of PLA in the printed square, the researchers analyzed the compressive strength of the 3D printed stent, regardless of whether HNTs were added. The stent has a higher strain ratio with HNTs (PLA + H and PLA + H + Zinc) and a higher average compressive modulus than the square without HNTs (PLA), indicating that the addition of HNTs only caused a slight but not significant increase in the elasticity and elasticity of the PLA. Compressive strength (Figure 6). Due to the limitation of the test equipment, there is no maximum applied force (200 N) after the stent is broken. Therefore, researchers cannot get complete compressed data. Judging from the current data, PLA has a tendency to enhance the compression performance of PLA+H+Zn, but the enhancement is not obvious.
in conclusion
In this study, the 3D printed square is composed of PLA and zinc-doped HNT, which can be used as a potential bone implant application. The porosity of this material is similar to that of human bone tissue. The unique FBS+NaOH+FBS sandwich coating is applied to the printed grid to enhance the hydrophilicity and promote cell adhesion and metabolism. Without the addition of exogenous osteogenic agents, the sandwich-coated PLA square can also induce osteoblasts to differentiate into osteoblasts. In addition, the outer coating of gentamicin reduces the risk of infection without negatively affecting bone formation. The newly designed composite material PLA+H+Zn also has good mechanical strength and osteoinductive ability, and can be used as a candidate material for 3D printing of bone implants. In addition, the surface modification strategy used in this study can also be used for other 3D printing applications.
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