3D Printing of Lotus Root-Like Biomimetic Materials for Cell Delivery and Tissue Regeneration

Abstract

Biomimetic materials have drawn more and more attention in recent years. Regeneration of large bone defects is still a major clinical challenge. In addition, vascularization plays an important role in the process of large bone regeneration and microchannel structure can induce endothelial cells to form rudimentary vasculature. In recent years, 3D printing scaffolds are major materials for large bone defect repair. However, these traditional 3D scaffolds have low porosity and nonchannel structure, which impede angiogenesis and osteogenesis. In this study, inspired by the microstructure of natural plant lotus root, biomimetic materials with lotus root-like structures are successfully prepared via a modified 3D printing strategy. Compared with traditional 3D materials, these biomimetic materials can significantly improve in vitro cell attachment and proliferation as well as promote in vivo osteogenesis, indicating potential application for cell delivery and bone regeneration.

Keywords: biomimetic materials; cell delivery; lotus root‐like biomaterials; tissue regeneration.

Figure 1

The feasible applications and fabrication of lotus root‐like biomimetic materials inspired by lotus root microstructure. a) The schemata of the functions of lotus root microstructure and the same microstructure in lotus petiole (the inset). b) The schemata of the application in tissue regeneration of lotus root‐like biomimetic materials. c) Traditional 3D printer nozzle with simple shell structure, d) the embedded structure of the modified 3D printer nozzle inspired by lotus root microstructure and e) 3D printing process. f) The traditional 3D printing scaffolds packed by solid struts. g) The lotus root‐like biomimetic materials packed by struts with different numbers of channels.

Figure 2

Morphology regulation and control of the lotus root‐like biomimetic materials. a) Biomimetic materials of different chemical compositions (e.g., ceramics, metal, and polymer) with lotus root‐like structure can be prepared by the modified 3D printing strategy. b) 3D micro‐CT images of biomimetic materials with different shapes (i.e., cube, disk, and rod) and different numbers of channels (e.g., 4CSP representing four channels in one strut). The shapes of biomimetic materials can be well controlled by the predesigned programs for 3D printing. c) The number and channel size can be well controlled by designing corresponding nozzle with embedded structure.

Figure 3

Characterizations of biomimetic silicate‐based bioceramic (Akermanite, (AKT), Ca₂MgSi₂O₇) scaffolds with lotus root‐like microstructure. a) Optical microscope and b) SEM images show the lotus root‐like biomimetic structure. The materials are packed by struts (Ø1.5 mm) with different numbers of channels (Ø400–600 µm). c) SEM image for cross‐section showing that the hollow channels are completely open. d) SEM image for the inner surface microstructure of the channels. e) XRD analysis demonstrating the pure crystal phase of silicate‐based bioceramic scaffolds. f) Packing patterns (i.e., cross packing pattern, quartet close packing pattern, and hexagonal close packing pattern) have significant influence on the g) porosity and h) compressive strength of the lotus root‐like biomimetic materials. Porosity and mechanical property can be well controlled by predesign of packing pattern and number of channels. (n = 5, **P < 0.01, ***P < 0.001.)

Figure 4

BMSCs cultured in TSSP, 1CSP, 2CSP, 3CSP, and 4CSP‐AKT bioceramic scaffolds for different time periods. a,b) SEM images of BMSCs attached in the channels of biomimetic scaffolds after culturing for 3 d. b) BMSCs adhered on the scaffolds via numerous filopodia as shown by the yellow arrows. c–e) The CLSM images for the morphology and cytoskeleton of BMSCs on the surface of struts and channels in TSSP, 1CSP, 2CSP, 3CSP, and 4CSP scaffolds after culturing for 3 d. d) Surface magnified image and e) 3D image shows that BMSCs penetrated into channels and attached on the inner walls of channels. f) The amount of adhered BMSCs after 4, 8, 16, and 24 h culturing and g) the proliferation activity of BMSCs in different scaffolds after 1, 3, and 7 d of incubation respectively, detected by the CCK‐8 assay. The initial adhered cells and their proliferation activity enhanced with the increase of the channel numbers in the biomimetic scaffolds. (n = 6, **P < 0.01, ***P < 0.001.)

Figure 5

Characterizations of the lotus root‐like biomimetic scaffolds to enhance in vivo angiogenesis in rat muscle implantation and osteogenesis in rabbit calvarial defects. a) Fluorescence image of histological sections of biomimetic scaffolds stained with DAPI. b,c) The sections from microfil‐perfused samples were used to detect the new blood vessels, b) optical microscope image of 3CSP biomimetic scaffolds with blood vessels perfused by microfil, c) the magnified image of blood vessels (in blue) in the lotus root‐like structure. d) Typical 3D reconstruction micro‐CT images of the edges between materials and rabbit calvarial defects, and e) micro‐CT cross‐section images of rabbit calvarial defect regions (red for new bone tissues, green for materials). f) The undecalcified histological sections stained with Van Gieson's picrofuchsin, newly formed bone tissues (in red) can be well observed (blue arrows point to the new bone). g) Micro‐CT reconstruction analysis of the volume ratio of the newly formed bone to the defect regions (BV/TV) and histological morphometric analysis of the area of the newly formed bones in the whole defect regions at week 12. The 3CSP biomimetic materials showed significantly improvement in bone regeneration as compared to 1CSP and TSSP materials. (n = 6, *P < 0.05, **P < 0.01, and ***P < 0.001.)