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| Source： EngineeringForLife Avascular necrosis of the femoral head is a common disease of the hip joint. Due to damage and destruction of the blood supply of the femoral head, avascular necrosis of bone cells and bone marrow leads to structural changes and collapse of the femoral head. Recently, researcher Zhou Changchun from Sichuan University and his team used 3D printing technology to prepare a 3D printed porous Ti6Al4V reconstruction rod (called reconstruction rod) loaded with icariin (IC). The mechanical validity of the reconstructed rod was verified through finite element analysis. The loading of icariin was achieved by filling the icariin-containing thio-based hyaluronic acid hydrogel into the porous structure. The biological efficacy of the reconstituted rod was confirmed through in vitro cell experiments, showing that it can enhance MC3T3-E1 cell proliferation and promote cell adhesion and spreading. The therapeutic effect of the reconstructed rod was verified in vivo through an animal femoral head necrosis model. The results showed that the reconstruction rod promoted osteogenesis and neovascularization, leading to effective bony fusion between bone and implant. This study provides an innovative strategy for the treatment of early avascular necrosis of the femoral head.
The relevant research content was published in "Acta Biomaterialia" on August 1, 2023 under the title "Icariin-loaded 3D-printed porous Ti6Al4V reconstruction rods for the treatment of necrotic femoral heads".  Figure 1 3D model design and functional structural characterization of porous Ti6Al4V reconstructed rods The 3D model and solid structure of the optimally designed reconstruction rod are shown in Figure 1. The length of the reconstruction rod is 30 mm and consists of two parts: the front end and the back end. The front end is cylindrical, 20mm long and 5mm in diameter. It consists of a porous diamond unit that simulates human cancellous bone trabeculae, with a pore size of 660 μm and a porosity of 70%. The rear end is a solid rod-like structure with gradient changes in diameter. Its length is 10 mm, with an initial surface diameter of 3 mm and gradually increasing with a gradient of 5°. The surface of the reconstruction rod is covered with threads, the screw depth is 1 mm, and the thread pitch is 1.5 mm. At its tail, a groove structure matching the tool is designed, with a groove depth of 5 mm and a groove width of 0.5-2.3 mm. A cylindrical hole with a diameter of 2 mm was constructed through the reconstruction rod along the long axis.  Figure 2 Finite element model and equivalent stress distribution network diagram of each group Finite element analysis (FEA) was used to eva luate the mechanical effectiveness of the reconstructed rods, and the Von-Mises equivalent stress distribution was used as the output. When the load value is 3 times the body weight, in the healthy group before rod implantation, the equivalent stress value on the femoral head surface is in the range of 0-23.33 MPa, which is far lower than the yield strength of cortical bone (about 100 MPa) (Figure 2A ). The equivalent stress distribution of the reconstructed rod is in the range of 0~8.97MPa, which is much lower than the yield strength of the porous Ti6Al4V alloy (about 100 MPa) (Figure 2B). Sustained force has the potential to cause further extension of the fracture, ultimately leading to complete fracture of the cervical femur, resulting in loss of structure and function of the entire joint (Figure 2C). At the same time, the equivalent stress of implanted porous reconstruction rods was distributed within a safe range (Figure 2D). The maximum equivalent stress value of the reconstructed rod reaches 118.50 MPa, which exceeds the yield strength of the porous Ti6Al4V alloy, and there is a risk of yield deformation and fatigue fracture (Figure 2E). The equivalent stress concentrated on the reconstructed rod is also reduced below the yield strength to avoid yielding and fatigue-induced deformation and fracture (Figure 2F). The statistical histogram of the maximum equivalent stress value distribution of each group is shown in Figure 2G, and the results show the same trend.  Figure 3 Physical and chemical characterization of hyaluronic acid (HA-SH) hydrogel HA-SH is formed through a self-crosslinking reaction, forming a disulfide bond between sulfhydryl groups, and its chemical structural formula is shown in Figure 3A. The successful grafting of thiol groups was verified by 1H NMR, and the grafting rate was calculated to be approximately 34.5% (Figure 3B). The HA-SH hydrogel was extruded through a syringe into customized text, reflecting its injectable properties (Figure 3C). The gelation rate of HA-SH in each group was similar, with a transparent appearance and high purity, and the difference was not statistically significant (Figure 3D). In the swelling experiment, HA-L and HA-M reached swelling equilibrium faster than HA-H (Figure 3E), indicating that within a certain range, the higher the solute concentration, the stronger the water absorption capacity, and the higher the swelling ratio, the more It is not easy to achieve swelling equilibrium. In the degradation experiment, HA-H and HA-M reached degradation equilibrium faster than HA-L (Figure 3F), indicating that within a certain range, the lower the solute concentration, the faster the degradation rate and the worse the degradation resistance. The higher the quality loss ratio. The storage modulus reflects the elasticity of the material, while the loss modulus reflects the viscosity of the material. The viscoelasticity of HA-H is higher, while the viscoelasticity of HA-M and HA-L is lower (Figure 3G, H), which shows that HA-SH The viscoelasticity of a hydrogel is related to its concentration. The viscosity of HA-SH hydrogel decreases with the increase of shear frequency (Figure 3I), which has the characteristics of shear thinning, which is beneficial to its injection behavior.  Figure 4 Schematic diagram and morphological characterization of each component in the porous region of the reconstructed rod The icariin-loaded 3D printed porous Ti6Al4V reconstruction rod is based on Ti6Al4V alloy, and the pores in the porous area are filled with icariin-loaded HA-SH hydrogel. The schematic diagram of each component is shown in Figure 4A. The Ti6Al4V alloy prepared by 3D printing has a clearly visible and well-arranged porous structure (Figure 4B). HA-SH hydrogel has an irregular porous structure with a size distribution between tens to hundreds of microns, providing space for cell adhesion and tissue growth, and using body fluids as the medium to achieve intermolecular transport of nutrients and other substances. Conducive to the slow release of drugs (Figure 4C). IC powder is in the form of granules or rods with size distribution ranging from hundreds of nanometers to tens of micrometers (Figure 4D).  Figure 5 Cell viability and proliferation of MC3T31, 3, and 7 days cultured on Ti, T3-E1, and Ti-HA-IC scaffolds CCK-8 detection results showed that compared with blank and Ti, Ti-HA-IC showed both a higher number of cells (Figure 5A) and a faster proliferation rate (Figure 5B). The number of cells on the surface of Ti-HA-IC is relatively large and has a certain distribution pattern, which outlines the porous structure of the scaffold. On the 7th day of co-culture, the cell density on the surface of both scaffolds increased significantly, and the distribution patterns were similar. In contrast, on the Ti-HA-IC surface, the number of cells was larger, the cells were more densely distributed, and the porous structure outlined by the cells was clearer (Figure 5C), proving that both scaffolds are cytocompatible.  Figure 6 Cell distribution morphology of MC3T3-E1 cultured on Ti and Ti-HA-IC scaffolds on day 7 Figure 6A-F are CLSM images of cells in different density distribution areas and semi-quantitative results of the average diffusion area of cells under phalloidin/DAPI staining. The results all show that the average diffusion area of cells on the Ti-HA-IC scaffold is higher than that on Ti stand. Figure 6G-L shows scanning electron microscope (SEM) images of cells in different surface areas and semi-quantitative results of the average cell diffusion area. The results show that the average cell diffusion area on the Ti-HA-IC scaffold is higher than that on the Ti scaffold (white arrows point to representative cytoskeletal structure). In summary, when IC is loaded into the scaffold at a certain concentration, the scaffold exhibits cytocompatibility and promotes the adhesion and spreading of MC3T3-E1 cells on the scaffold.  Figure 7 In vivo and in vitro modeling of avascular necrosis (AVN) and study of its characteristics In in vitro experiments, the femoral head showed obvious frostbite characteristics after modeling, and could not recover for a long time after heating again, suggesting the existence of an irreversible necrosis process (Figure 7A). In the in vivo experiment, HE staining showed that the bone was clear, the structure was dense and orderly, and the bone cell phenotype was normal before modeling (Figure 7B). After modeling, the bone structure was dissolved and the trabecular bone was disorganized (Figure 7C). MRI signal abnormality is a sensitive feature for the early diagnosis of osteonecrosis. The high signal and low signal of the femoral head are mixed, showing a "graph pattern" and a "double line sign" (Figure 7D). These results confirmed the occurrence of AVN. X-rays show that the femoral head basically shows a tendency to aggravate necrosis without implanting the reconstruction rod; after implanting the reconstruction rod, the overall shape and physiological structure of the femoral head does not change significantly, the joint space is clear, and the gray value density is evenly distributed. There were no obvious irregular changes and no obvious characteristics of AVN such as fragility fractures, hyperplasia and collapse (Figure 7E). Without the reconstruction rod implanted, typical necrosis of the femoral head was observed at 16 weeks after surgery; in contrast, the femoral head with the reconstruction rod was shiny, normal in shape, and highly spherical (Figure 7F).  Figure 8 Using Micro-CT to observe the femoral head bone volume and bone morphology 16 weeks after surgery As shown in Figure 8A, the 3D model of the femoral head in each group (including the reconstruction rod) was reconstructed, and the macroscopic shape (yellow) was observed. The 3D model of the ROI bone in each group (red hemispherical area) was reconstructed, and the outline structure (gray) was observed. Bone volume fraction (BV/TV) can directly reflect changes in bone mass. The results show that the value of the Ti-HA-IC group is similar to that of the healthy group, while the value of the Ti group is slightly lower, and the value of the control group is the lowest (Figure 8B ). Bone surface area density (BS/TV) can indirectly reflect changes in bone mass. The results showed that the value in the Ti group was close to the value in the healthy group, while the value in the control group and Ti-HA-IC group decreased significantly (Figure 8C). In order to further analyze the lower BS/TV value in the Ti-HA-IC group, representative coronal gray value images in each group were collected for comparison. Compared with other groups, the bone density in the Ti-HA-IC group was The distribution is denser, the gaps between bone trabeculae are smaller, and there is no obvious low-density area (Figure 8D). At the same time, select the center position of the femoral head at the front end of the reconstruction rod in each image, calculate the average gray value of the specified area (in the red circle), and draw it into a line graph. Among them, the Ti-HA-IC group has the best numerical calculation result. (Figure 8E). The 3D models of the blood vessels around the femoral head in the Ti group and the Ti-HA-IC group were reconstructed through angiography. The blood vessels in the Ti-HA-IC group were densely distributed and numerous. The main blood vessels were thicker and the new capillaries were smaller (Fig. 8F). Through comparison of statistical values, the volume and area of blood vessels around the femoral head in the Ti-HA-IC group were larger than those in the Ti group (Figure 8G). This shows that the sustained release of IC in the Ti-HA-IC group simultaneously promotes the repair and regeneration of bones and blood vessels, and the formation of blood vessels further accelerates bone formation, providing a synergistic treatment for AVN. In summary, the 3D printed porous Ti6Al4V reconstruction rod loaded with icariin has the best effect on the repair and treatment of AVN. 图片 Figure 9 HE staining and fluorescence methods were used to detect new bone formation and trabecular bone morphology of the femoral head at 16 weeks after surgery. The results of HE staining are shown in Figure 9A and B. The representative images of the contact position (or equivalent position) between the femoral head and the reconstruction rod are shown in Figure 9A. The representative images of the front position (or equivalent position) of the reconstruction rod are shown in Figure 9A and B. 9B. The trabecular bone has a thicker shape, a higher overall distribution density, and a block-like distribution characteristic. Semi-quantitative results showed that there was a certain degree of difference in bone area proportions between groups (Figure 9C). In the Ti group and Ti-HA-IC group, there was no obvious bone loss overall. The semi-quantitative results showed that compared with the healthy group, the bone area ratio in the control group was significantly reduced. There was no significant difference in the Ti group and the Ti-HA-IC group. The bone area ratio increased significantly (Fig. 9D). These results indicate that without timely intervention and effective treatment, the scope of femoral head necrosis will gradually increase. The difference in new bone growth between the control group and the Ti-HA-IC group was compared by fluorescent labeling technology (Figure 9E). In the image, the fluorescence of the calcein label is green, the tetracycline hydrochloride label is red, and the consistency of the two is orange. In the control group, the two fluorescence trajectories overlapped significantly, indicating that new bone growth slowed or even stopped. In the Ti-HA-IC group, the two fluorescence tracks were clearly separated, indicating that new bone was still growing under the action of the icariin-loaded 3D printed porous Ti6Al4V reconstruction rod. In summary, this study developed a 3D printed porous Ti6Al4V reconstruction rod loaded with icariin, which provides mechanical support for maintaining the functional structure of the femoral head, releases IC, promotes bone repair and regeneration, and achieves effective integration between the body and the implant. Integrate. Finite element analysis results show that the implanted reconstruction rod can transfer bone compression, effectively support the load and maintain the structure and function of the femoral head. In vitro cell experiments showed that IC-loaded scaffolds promoted the proliferation of MC3T3-E1 cells and facilitated cell adhesion and spreading on the scaffolds. In vivo animal experimental results show that the reconstruction rod implanted in the early stage of AVN not only provides mechanical support for the femoral head, but also provides a favorable environment for the growth of new bone, thus forming effective interface integration. In addition, the sustained and slow release of IC further promotes the regeneration of new bone and blood vessels, leading to an increase in femoral head bone mass and bone density, which has a positive effect on the treatment of necrosis. Therefore, this study provides a potential strategy for early treatment of AVN. Article Source: https://doi.org/10.1016/j.actbio.2023.07.057 |
