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. 2024 Apr 25;16(18):22887–22899. doi: 10.1021/acsami.4c01520

Lithium-Doped Titanium Dioxide-Based Multilayer Hierarchical Structure for Accelerating Nerve-Induced Bone Regeneration

Qianqian Zhang †, Shuting Gao ‡, Bo Li §, Qian Li †, Xinjie Li †, Jingyang Cheng ∥, Zhenjun Peng ⊥, Jun Liang #, Kailiang Zhang †,*, Jun Hai ∇,*, Baoping Zhang †,*
PMCID: PMC11082843  PMID: 38663861

Abstract

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Despite considerable advances in artificial bone tissues, the absence of neural network reconstruction in their design often leads to delayed or ineffective bone healing. Hence, we propose a multilayer hierarchical lithium (Li)-doped titanium dioxide structure, constructed through microarc oxidation combined with alkaline heat treatment. This structure can induce the sustained release of Li ions, mimicking the environment of neurogenic osteogenesis characterized by high brain-derived neurotrophic factor (BDNF) expression. During in vitro experiments, the structure enhanced the differentiation of Schwann cells (SCs) and the growth of human umbilical vein endothelial cells (HUVECs) and mouse embryo osteoblast progenitor cells (MC3T3-E1). Additionally, in a coculture system, the SC-conditioned media markedly increased alkaline phosphatase expression and the formation of calcium nodules, demonstrating the excellent potential of the material for nerve-induced bone regeneration. In an in vivo experiment based on a rat distal femoral lesion model, the structure substantially enhanced bone healing by increasing the density of the neural network in the tissue around the implant. In conclusion, this study elucidates the neuromodulatory pathways involved in bone regeneration, providing a promising method for addressing bone deformities.

Keywords: lithium, titanium dioxide, multilayer hierarchical structure, neurogenesis, bone regeneration

1. Introduction

Clinical applications of bone tissue engineering have consistently faced challenges related to bone defects.1,2 Traditional bone tissue engineering focuses on vascularization and other biological processes that are directly related to bone formation.3,4 Different from traditional engineering, which typically focuses on a single osteogenic signaling pathway, successful bone regeneration relies heavily on the presence of a functional nerve supply. This supply provides trophic support to bone cells, regulates blood flow, and modulates the inflammatory response.5 However, current materials for bone tissue regeneration exhibit only one-sided functionality, resulting in a gap in nerve-induced osteogenesis. Moreover, in clinical settings, there is a growing demand for multifunctional materials that can promote both osteogenesis and angiogenesis while supporting nerve growth. To meet these high standards, biomaterials with a wide range of properties are needed.6,7

The theoretical and mechanistic foundations of sensory neuron-induced bone tissue generation have been established.8,9 In response to initial nerve signals, several neuropeptides or neurotransmitters released by nerve cells—including neuropeptide Y (NPY), calcitonin gene-related peptide (CGRP), and brain-derived neurotrophic factor (BDNF)—influence bone metabolism and bone remodeling.10−13 BDNF and tyrosine kinase receptor B (TrkB) were found to be involved in vascular formation and osteogenic processes during human fracture healing.14 In one study, sympathetic nerve activity in the central nervous system could be controlled through the regulation of bone homeostasis via the activation of cAMP-response element-binding protein (CREB) signaling in the hypothalamus.15 Although the above findings suggest that the production of neuropeptides is a strong candidate for the regulation of bone regeneration, there is a lack of consensus on the development of new strategies for implant materials based on bone–nerve crosstalk. Surface topography, particularly geometric cues, offers advantages in stimulating both bone and nerve regeneration by altering the adhesion and morphology of target cells and controlling intracellular signal transduction.16,17 This approach allows for creating customized macro- and microstructures and controlling the interaction of cells with their environment.18,19 Engineering titanium (Ti) surfaces with microgeometric patterns is important for promoting bone regeneration and inducing osteogenic differentiation.20

Lithium (Li) is capable of enhancing both osteogenesis and nerve regeneration, thereby exhibiting a dual functionality. It is widely utilized in trauma and long-term neurodegenerative treatment owing to its explicit role in nerve regeneration and the facilitation of neurotransmitter transmission.21−23 Studies have shown that Li increases BDNF expression by triggering several signaling pathways, including the cyclic adenosine phosphate (cAMP) signaling pathway.24,25 Furthermore, Li exists as a trace element in human bone tissue. It promotes bone formation by activating the Wnt signaling pathways and stabilizing β-catenin by preventing the enzyme activity of glycogen synthase kinase-3 (GSK-3β).26−29 Although some studies have investigated the impacts of Li on bone regeneration and the growth of Schwann cells (SCs), there is a scarcity of research on the mechanisms involved in creating composite structures using Li and micropatterns for neural osteogenesis.30−32

In the present study, through microarc oxidation (MAO) and alkali-heat treatment, we developed a Li-doped multilayer structure on a titanium dioxide construct embedded with calcium phosphorus (Scheme 1). The physical and chemical properties of the scaffolds were analyzed. The characterization results revealed that the structure exhibited excellent biosafety. The structure effectively attracted osteoblasts and SCs, significantly increasing osteogenic and neurogenic differentiation. The structure also effectively regulated vascularization in in vitro experiments using relevant biological materials. Additionally, constructs cocultured with 3T3-SC significantly enhanced nerve-induced bone regeneration. Moreover, this construct successfully established an in vivo microenvironment with suitable spatial characteristics for further innervation of bone regeneration. In vivo and in vitro studies showed that the mechanism of action involves promoting bone and nerve regeneration by activating the Wnt/GSK-3β/β-catenin pathway. With the advancement in the understanding of neurovascular coupling in bone homeostasis and regeneration, new biomaterial-based therapies aimed at promoting nerve–bone interactions will substantially enhance outcomes in bone repair management and alleviate patients’ pain and suffering.

Scheme 1. Diagrammatic Illustration of a Multilayer Hierarchical Li-Doped Titanium Dioxide Structure and Its Application in Nerve-Induced Bone Regeneration.

Scheme 1

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2. Results and Discussion

2.1. Characterization of Li-Doped Multilayered Hierarchical Coatings

Through MAO, we achieved a uniform, dense, dark brown coating on a Ti alloy surface. This coating was subjected to a straightforward alkali-heat treatment with LiOH, and a bright brown, dioxide-based multilayer hierarchical structure was obtained. Figure 1A–F show scanning electron microscopy (SEM) images of uncoated and coated materials. The MAO coating surface exhibited tiny pores that resembled small volcanoes, each with a pore diameter of ∼3 μm. Figure 1D,E displays the surface structure of the MAO@Li0.05 group. In contrast to the MAO group, the Ti sheets in the MAO@Li group exhibited nanofine lines and fine needlelike structures. Our multilayer structure was constructed as a nanorod, needlelike structure on the Ti surface through a simple alkaline thermal reaction, grown directly on the Ti sheet without the need for a complex assembly process. In tissue engineering, a multilayer scaffold can be engineered to include layers that provide mechanical support, mimic the extracellular matrix, and promote cell adhesion and proliferation, thereby enabling interaction with biological systems in a more biomimetic and effective manner.33−35

Figure 1.

Figure 1

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Characterization of Li-doped multilayered hierarchical coatings. (A), (B) SEM images of MAO coating. (C) Cross-sectional SEM image of MAO coating. (D), (E) SEM images of MAO@Li0.05 coating. (F) Cross-sectional SEM image of MAO@Li0.05 coating. (G), (H) HR-TEM images of MAO@Li0.05 coating. (I) Selected area electron diffraction (SAED) image of MAO@Li0.05 coating. (J) (K) STEM images of MAO@Li0.05 coating. (L) AFM image of MAO@Li0.05 coating.

Furthermore, the cross-sectional micromorphology revealed a tight bond between the coating and the Ti alloy substrate, and the coating was uniformly distributed (Figures 1C,F and S1B). The MAO coating had a thickness of 3.2 ± 0.2 μm, whereas the coatings of the MAO@Li group had a thickness of approximately 2.6 ± 0.1 μm (Figure S2). The results demonstrate that after treatment with alkali and heat, the coatings of MAO@Li groups became thinner. This is attributable to the following chemical reactions that occurred during this process:36 TiO2 + OH– → HTiO3–, nCa2+ + nHTiO3– + nOH– → [Ca–Ti–O3]n-nH2O, nLi+ + nHTiO3– + nOH– →[Li2–Ti-O3]n-nH2O. The reactions can be interpreted as follows: (a) dissolution of Ca and P, (b) formation of negatively charged HTiO3–, (c) redeposition of Ca and Li, and (d) final deposition of Ca–Ti–O and Li–Ti–O compounds on the material surface. These reactions, modulated by factors such as time and temperature, account for the coexistence of the porous structure with fine-grained and needlelike structures and the reduction in coating thickness. The structure of the Li-doped coatings was further revealed by representative transmission electron microscopy (TEM) images (Figure 1G). Polycrystalline Li2O and TiO2 were detected in high-resolution transmission electron microscopy (HRTEM) images and associated electron diffraction patterns (Figure 1H,I). The index planes, specifically 111 and 220, corresponded to phases of Li2O,37 indicating successful Li doping into the coating, with some of the Li present as Li2O. The scanning transmission electron microscopy (STEM) images of the MAO@Li0.05 group (Figure 1J,K) revealed the major constituents of the coating, which included Ti, O, Ca, and P. However, Li ions may not be readily detectable through the energy-dispersive X-ray spectroscopy assay. Figure 1L illustrates the surface morphology of the MAO@Li0.05 group, and Figures S3 and S4 provide data on the change in the coating roughness. Atomic force microscopy (AFM) height maps further elucidate the changes in roughness after the alkali–heat treatment.

According to the X-ray diffraction (XRD) patterns of the coatings in each group (Figure 2A), the predominant components of both the MAO and MAO@Li groups were the anatase and rutile phases of TiO2. The alkali–heat treatment increased the anatase phase, consistent with the findings of previous studies.38 The increase in the anatase phase in the MAO@Li group possibly enhanced osteogenic activity or resulted in an increase in hydroxyl groups on the anatase surface.39 The MAO@Li group also exhibited a minor Li2O phase. The materials in both the MAO and MAO@Li groups were primarily composed of Ti, Ca, P, and O, with the MAO@Li group also containing trace amounts of Li, as indicated by the full-spectrum X-ray photoelectron spectroscopy (XPS) results (Figure 2B). According to the XPS fine spectra of the Ti 2p, Ca 2p, P 2p, and Li 1s peaks in the MAO group (Figure S5) and MAO@Li0.075 group (Figure 2C–F), both groups of coatings exhibited Ti 2p 3/2 orbital binding energies of 458.5 eV and Ti 2p 1/2 orbital binding energies of 464.4 eV, corresponding to Ti4+. Ca2+ was present on the coating’s surface, as evidenced by the Ca 2p 3/2 binding energy of 347.2 eV and the Ca 2p 1/2 binding energy of 350.8 eV. The unimodal protuberance P 2p, corresponding to P5+ and situated at 133.04 eV, was consistent with the P–O bond of PO43–. The decrease in the unimodal diffraction of P 2p in the MAO@Li0.075 group is attributable to P dissolution during alkali–heat treatment. Li 1s appeared as a single peak at 52 eV in the MAO@Li0.075 group, indicating that the chemical valence of Ca, P, Ti, and other key elements was unaffected by the alkali–heat treatment with LiOH.

Figure 2.

Figure 2

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Analysis of the physical and chemical characteristics of various coating compositions. (A) XRD patterns of all the samples. (B) XPS survey spectrum of all the samples. (C–F) High-resolution XPS spectra of Ti 2p, Ca 2p, P 2p, and Li 1s in MAO@Li0.075. (G) Water contact angle image display. (H) Quantitative analysis of surface hydrophilicity changes in different coatings. (I) Analysis of the released and accumulated Li ions in different coatings. (J) Three-dimensional illustrations of wear marks from various friction groups. The data of Figure 2H was analyzed using one-way ANOVA and presented as the mean ± SD *p < 0.05, ** p < 0.01, ***p < 0.001 and * represent statistical significance between the indicated groups.

However, the composition of some elements changed slightly after the alkali–heat treatment owing to ion exchange induced by chemical etching. Supplemental Table 1 compares the elemental changes in the MAO@Li0.075 group and the MAO group. Li content increased from 0% in the MAO group to 15.77 ± 0.86% in the MAO@Li0.075 group, confirming the successful incorporation of Li into the coating during the alkali–heat treatment. As depicted in Figure 2G, Li functionalization increased the wettability of the surface: the contact angles for the Ti, MAO, and MAO@Li0.05 groups were 81.4° ± 4.5°, 33.3° ± 4.5°, and 9.8° ± 0.7°, respectively. The average hydrophilic angle for the MAO@Li group was 11.1° ± 0.7°, attributable to the introduction of Ti–OH groups following alkali–heat treatment; this significantly improved the hydrophilicity of the implants, consistent with trends reported in the literature.40 The MAO@Li group exhibited significantly lower water contact angles than the Ti and MAO groups (Figure 2H). The improved wettability and roughness of the modified coating surface have the potential to promote osteogenesis in subsequent applications.41,42

According to the inductively coupled plasma-optical emission spectrometry (ICP-OES) results presented in Figures 2I and S6A,B, the release of Ca, P, and Li ions decreased gradually after 7 days and a small number of ions were still released after 32 days. The release of Li ions enables subsequent biological effects, as Li+ has been demonstrated to possess multifunctional and therapeutic properties, promoting both cell proliferation and angiogenesis.43,44 The friction coefficients for the Ti, MAO, and MAO@Li0.05 groups, with an 8.5 N loading, are displayed in Figure S6C. At the start of the experiment, the modified group exhibited a friction coefficient that was higher than that of the Ti group. However, at the end of the experiment, both groups exhibited roughly equal coefficients. The three-dimensional (3D) diagram of the wear marks of each group following the friction test is displayed in Figure 2J; the MAO@Li0.05 group exhibited a minimal wear depth at 34 μm. Further, the semiquantitative analysis of wear depth among the Ti, MAO, and MAO@Li0.05 groups were 40.87 ± 1.21 μm, 52.67 ± 2.52 μm, and 32.27 ± 2.05 μm, respectively (Figure S6D), underscoring the efficacy of the Li-doped MAO coating in reducing friction and minimizing wear.45

2.2. Biocompatibility Test of Multilayer Coatings

Cell counting kit-8 (CCK-8) was used to calculate the proliferative growth rate of SCs cultured on various samples (Figure 3A). Overall, the MAO@Li group exhibited a cell density higher than those of the Ti and MAO groups. The SEM morphology and Calcein-AM staining of the cultured cells on the various samples were examined to confirm this result. In addition, the MAO@Li group exhibited a higher cell density, more pseudopodia, and more pronounced cell spreading and expansion than the Ti and MAO groups (Figures 3B–D). The early morphological changes of SCs on the material surface are depicted in Figure 3D. The MAO and MAO@Li groups exhibited more cells, filamentous pseudopodia, and plate pseudopodia, as well as a more extensive, 3D, and cross-linked cellular structure, which is a typical indication of cell differentiation and maturation. The MAO@Li0.05 group exhibited a pronounced manifestation of this phenomenon and the characteristic morphology of bipolar spindle cells. After 48 h of culturing, skeletal morphology and the semiquantitative analysis demonstrated that the MAO@Li0.05 group exhibited a higher average fluorescence intensity for F-actin and DAPI than the other groups (Figure 3E and S7). Overall, these findings underscore the outstanding biocompatibility of the prepared Li-doped coating.

Figure 3.

Figure 3

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Biocompatibility and the impact of different coatings on Schwann cells in vitro. (A) Proliferation of SCs in different coatings by CCK-8 assay. (B) Calcein-AM staining and fluorescent images showing the arrangement of SCs on different coatings after 3 days. (C) Fluorescence intensity of Calcein-AM staining for SCs on different coatings. (D) SEM images showing the morphology of SCs on different coatings. (E) Skeletal morphology of SCs on the coating surface of each group. The rhodamine phalloidin-labeled cytoskeletons and DAPI-labeled nucleus are shown in red and blue, respectively. The data of Figure 3A,C were analyzed using one-way ANOVA and presented as the mean ± SD *p < 0.05, ** p < 0.01, ***p < 0.001, and *represent statistical significance between the indicated groups.

2.3. Modulation of Neurogenic Osteogenesis by the Wnt/GSK-3β/β-Catenin Pathway

Associated genes were examined to determine the effects of different coatings on the osteogenesis potential of MC3T3-E1 cells and the growth of SCs. RT-PCR analysis revealed that compared with the other groups, the MAO@Li0.05 group showed increased expression of genes involved in nerve growth in SCs, including BDNF, glial cell-derived neurotrophic factor (GDNF), CREB, myelin protein zero (MPZ), and peripheral myelin protein-22 (PMP22). These results highlight the pronounced expression of nerve growth factors in the MAO@Li0.05 group. For instance, GDNF, which is one of the primary cues used by SCs to direct axons into distal neural stumps, promotes the growth of SCs.46 The expression of BDNF mRNA in the MAO@Li0.05 group was nearly seven times that in the Ti group (Figure 4A(1)–(5)).

Figure 4.

Figure 4

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Expression of genes and protein in SCs, MC3T3-E1, and HUVECs cell lines. (A) RT-PCR analysis of the relative mRNA expression of genes related to osteogenesis, angiogenesis, and neurotrophins. (1–5) Relative neurotrophins gene (BDNF, GDNF, MPZ, PMP22, and CREB) expression in SCs on surface of the materials. (6–7) Relative osteogenesis gene (GSK-3β and BMP-2) expression in MC3T3-E1 on surface of the materials. (8) Relative angiogenesis gene VEGF expression in HUVECs on surface of the materials. (B) Protein levels and quantitative analysis of the expression levels of proteins associated with neurotrophins, osteogenesis, and angiogenesis. The data of Figure 4A,B were analyzed using one-way ANOVA and presented as the mean ± SD *p < 0.05, ** p < 0.01, ***p < 0.001, and * represent statistical significance between the indicated groups.

After the MC3T3-E1 cells were cultured on the coatings, BMP-2 mRNA expression matched that observed in SCs, whereas GSK-3β mRNA expression decreased, showing that the expression of GSK-3β mRNA was effectively suppressed (Figure 4A(67)). Additionally, Figure 4A(8) demonstrates that vascular endothelial growth factor (VEGF), the main mediator of pro-angiogenesis, was markedly upregulated in the MAO@Li0.05 group,47,48 indicating that our final coating possessed certain vascularization abilities. The release of VEGF directly regulates osteoblast and osteoclast differentiation and participates in bone remodeling. VEGF acts as a chemotactic molecule, attracting endothelial cells to bone tissue.49 The same trend was also evident in the further Western blotting results (Figure 4B). To further explore the potential role of the developed Li-doped material in nerve-induced bone regeneration, we evaluated the effect of the material on SCs and MC3T3-E1 cells. RT-PCR results confirmed that the Li-doped structure stimulated SCs to secrete BDNF in large quantities. This is attributable to the activation of CREB, which facilitates the transcription of crucial proteins, particularly BDNF, for activity-dependent plasticity.24,50 A previous study indicated that VuPAM-mediated inhibition of GSK-3β increased the phosphorylation of CREB.51 Our experiments demonstrated that Li ion-mediated inhibition of GSK-3β induced the CREB activation of downstream molecules. The initiation of this series of signals enables osteogenic differentiation in subsequent coculture systems.

Peripheral nerve repair involves the transplantation of bone marrow mesenchymal stromal cells (BMSCs) cocultured with SCs, as BMSCs can transform into SC-like cells.52 We constructed a coculture model to determine whether the Li-doped coating enhances the osteogenic potential of MC3T3-E1 cells via the sensory nerve (Figure 5A). To simulate SC-mediated nerve–bone crosstalk, the supernatant from SCs from the Ti, MAO, and MAO@Li0.05 groups was extracted to create a conditioned medium.

Figure 5.

Figure 5

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Results of osteogenic property studies in vitro. (A) Schematic illustration of the coculture model for neurogenic differentiation of MC3T3-E1. (B) Alizarin red staining (ARS) of the MC3T3-E1 after osteogenic induction for 28 days. (C) Skeletal morphology of MC3T3-E1 on the coating surface of each group. The rhodamine phalloidin-labeled cytoskeletons and DAPI-labeled nucleus are shown in red and blue, respectively. (D) Quantitative analysis of ALP activity on day 7 and ARS on day 28. (E) Semiquantitative analysis of MC3T3-E1 cytoskeleton staining on the surface of materials in each group. The data of Figure 5D,E were analyzed using one-way ANOVA and presented as the mean ± SD *p < 0.05, ** p < 0.01, ***p < 0.001, and * represent statistical significance between the indicated groups.

The results revealed a gradual increase in the deposition of calcium nodules in both the MAO and MAO@Li0.05 groups, indicating mineralization in MC3T3-E1 cells (Figure 5B). A semiquantitative analysis of Alizarin red staining further revealed that after 28 days, the MAO@Li0.05 group exhibited higher mineralization than the control group (Figure 5D). Activity quantification was employed to assess alkaline phosphatase (ALP) levels as an early marker of osteogenesis.53 ALP expression revealed a consistent pattern. Notably, the coculture group generally exhibited better outcomes in terms of osteogenic capacity than the single-culture group. Furthermore, cells in the coculture group displayed a typical elongated morphology, with actin fibers arranged in an orderly manner and exhibiting greater intensity (Figure 5C). Conversely, cells from the control group were rounded, with a disordered cytoskeleton and lower intensity. Moreover, the MAO@Li0.05 group displayed the highest average fluorescence intensity for both the cytoskeleton and nucleus in the coculture group (Figure 5E). These observations suggest that under coculture conditions, the Li-doped coating surfaces may induce cytoskeletal remodeling. Our current findings corroborate previous research, indicating that high BDNF mRNA expression in the MAO@Li0.05 group within the coculture setup can activate the Wnt/GSK-3/β-catenin signaling pathway during bone regeneration (Figure S8).

2.4. Performance Enhancement of Li-Doped Coating through Neurogenic Osteogenesis In Vivo

The developed Li-doped material was implanted into the tibiae of rats for 4 weeks to confirm its ability to stimulate osteogenesis and nerve fiber regeneration in vivo. The bone–implant bond in all groups remained stable and intact 4 weeks after implantation (Figure S9B). Microcomputed tomography imaging (Figure 6A) revealed that the newly formed bone occupied the implant’s bone interface and integrated effectively. Compared with the Ti and MAO groups, the MAO@Li0.05 group exhibited a more mature bone trabecula, and a greater amount of new bone occupied the implant–bone interface. Furthermore, compared with the Ti and MAO groups, the MAO@Li0.05 group exhibited higher values of bone volume/total volume (BV/TV), bone mineral density (BMD), and trabecular thickness (Tb.Th), along with lower values of trabecular separation (Tb.Sp). This indicates that the newly formed bones surrounding MAO@Li0.05 exhibited a significantly greater level of maturation and density than the other groups (Figure 6B). This in vivo observation was consistent with the in vitro results. Hematoxylin and eosin (H&E) staining (Figure 6C) confirmed the presence of a significantly greater new bone formation around the implants in the MAO@Li0.05 group than in the Ti and MAO groups 4 weeks after implantation. These findings demonstrate the effectiveness of the Li-doped Ti alloy implants in stimulating bone regrowth.

Figure 6.

Figure 6

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Performance enhancement of Li-doped coating through neurogenic osteogenesis in vivo. (A) Three-dimensional reconstruction of the implants for the three groups 4 weeks after surgery. (B) Quantitative analysis of the femoral by reconstruction. (C) H&E staining to evaluate osteogenesis associated with the various implants. (D) Immunohistochemistry and quantitative analysis of myelin nerve structures around the rat tibial implant, with positive results for both NF and NPY markers. The data of Figure 6B,D were analyzed using one-way ANOVA and presented as the mean ± SD *p < 0.05, ** p < 0.01, ***p < 0.001, and * represent statistical significance between the indicated groups.

The dark brown structure depicted in Figure 6D is a positive myelin nerve fiber structure. Myelin nerve structures occurred along the edge of the implant fossa, around the bone marrow cavity, within the spaces between the bone trabeculae, and in the periosteum of the rat tibial implant. These nerve structures tested positive for both nerve fibers (NF) and NPY.54,55 According to the semiquantitative analysis, 4 weeks after implantation, the MAO@Li0.05 group exhibited significantly higher expression area ratios of NF and NPY than the Ti and MAO groups. These findings underscore the material’s excellent potential to stimulate neurogenic bone regeneration and effectively promote the growth of peripheral nerve fibers in vivo. Major organs (liver, brain, spleen, heart, kidneys, and lungs) were collected from each group of rats for H&E staining (Figure S10). The results showed no adverse effects on the overall health of the rats, indicating the outstanding biocompatibility and biosafety of the coating.

Despite the contributions of this study, it has some limitations. First, the alkali thermal reaction used in material preparation did not thoroughly meet the requirements for stable release. Second, the advantages of the micronano multilayer hierarchical structure of the Li-doped material were not highlighted by the mechanisms of biological functions. Further investigation is needed to determine whether neurogenesis or angiogenesis is the primary influencing factor in osteogenesis for neurovascular coupling in bone homeostasis and regeneration. This can be achieved through a series of knockout experiments such as knocking out the gene level of VEGF and observing the effect of lithium elements. Our future work can address these challenges by optimizing the Li-doped method and comparing the biofunctions with those of the traditional coating structure.

3. Conclusion

Bone tissue is densely innervated by sensory and sympathetic nerves, and neurogenesis plays a crucial role in the bone regeneration process by secreting neurotransmitters, neuropeptides, axon guidance factors, and neurotrophins. In this study, we develop a Li-doped multilayer hierarchical structure based on a Ti substrate to promote innervation and accelerate bone regeneration. The Li-doped material possesses excellent biocompatibility and can significantly enhance the neural differentiation of SCs and osteogenic differentiation of MC3T3-E1 and promote angiogenesis in HUVECs in vitro. Notably, the Li-doped multilayer hierarchical structure activates sensory innervation in a rat distal femoral lesion defect model and enhances bone regeneration and remodeling. Our findings suggest that the Li-doped multilayer hierarchical structure creates a favorable microenvironment for nerve growth and bone regeneration, offering promising clinical applications to expedite and improve bone repair treatments in the future.

4. Experimental Section

4.1. Preparation of Composite Coatings

After being ground from 800 to 2000 mesh, the Ti-6Al-4 V samples, with a diameter of 24 mm and thickness of 1 mm (Baotian Group, China), were successively immersed in acetone solution, absolute ethanol solution, and ultrapure water for 15 min each, accompanied by sonic oscillation. For the MAO process, a combined solution of sodium dihydrogen (0.04 mol/L) phosphate and calcium acetate (0.01 mol/L) was used in direct current mode with a constant voltage. The temperature of the electrolyte was kept constant at 25 °C by a cooling system. Additional parameters for this process were a frequency of 500 Hz, a duty cycle of 10%, an oxidation voltage of 380 V, and an oxidation time of 10 min. After MAO, the titanium sheets were submerged in lithium hydroxide solutions (0.025, 0.05, and 0.075 mol/L) for 12 h at 100 °C.

4.2. Materials Characterization

Scanning electron microscopy (SEM, Thermo Fisher Apreo S, USA) was used to observe the microscopic morphology of the surface and cross section of the samples, and the thickness of the coating was measured by using a thickness gauge (ElektroPhysik MiniTest 1100; Cologne, Germany). The microstructure of the layer was examined using TEM (ElektroPhysik), and the topography and roughness of the sample surface were measured by AFM (Bruker NanoWizard V BioScience AFM, USA). Phase analysis of the samples was conducted using an X-ray diffractometer (XRD, D/Max-2400; Rigaku, Japan). An X-ray photoelectron spectrometer (XPS, Escalab 250Xi, Thermo Fisher, USA) was used to analyze the composition, proportion, and valence state changes of the elements in the coating. Surface wettability was evaluated by measuring the water contact angle on the surface of each group of materials using a contact angle meter (DSA100, Kruss, Germany). The release of Ca, P, and Li ions in the sample soaking solution was detected by using inductively coupled plasma-optical emission spectrometry (ICP-OES, Jena, Germany). Finally, the tribological properties of different groups of materials under artificial saliva lubrication were measured using a reciprocating tribometer (UMT TriboLab, Bruker, USA).

4.3. In Vitro Biocompatibility Test

The cell lines in this study included rat Schwann cell lines (SCs) (Procell, I CL-0199), MC3T3-E1 (ATCC, CRL-2595), and HUVECs (ATCC, CRL-1730). All cells were cultured in high-glucose DMEM containing 10% fetal bovine serum (FBS) and MEM-α containing 10% FBS. They were incubated at 37 °C under an atmosphere containing 5% carbon dioxide. Cell proliferation was assessed using CCK-8 (K1018; APExBIO, USA). Samples of each group were placed into 12-well plates, with 1 × 104 SCs inoculated in each well, and cultured for 1, 3, and 5 days. The optical density value of the samples was measured using a microplate reader (Multiskan Spectrum, Thermo Fisher Scientific, USA) at a wavelength of 450 nm. To evaluate live cells and to assess cell adhesion, Calcein-AM (C2012–0.1 mL, Beyotime, China) staining was employed. SCs were seeded on the surface of each group at a density of 1 × 104 cells/mL and cultured for 3 days. Then, the cells were incubated in the Calcein-AM working solution (stains living cells green) for 15 min at room temperature. The surface of materials with cell attachment was inverted and imaged by a fluorescence microscope (CKX53, Olympus, Japan). Then, we randomly selected and analyzed the fluorescence intensity in several fields on material surfaces via the ImageJ software (v1.8.0, NIH, USA).

Cytoskeletal organization was assessed in each group using immunofluorescence (IF) imaging. For this process, cells were seeded onto the samples from each group at a density of 1 × 104 SCs cells/mL. After being cultured for 48 h, the cells were stained with Acti-stain 555 phalloidin conjugate solution (PHDH1, Cytoskeleton, Inc., USA) and 4′,6-diamidino-2-phenylindole (DAPI; D9542, Sigma-Aldrich, USA) to label the nucleus. Finally, the materials were transferred onto glass slides using an automated slide scanner (Slideview VS200, Olympus, Japan) to capture the fluorescence emanating from the stained cells.

4.4. Expression Analyses for Neurotrophins/Osteogenesis/Angiogenesis-Related Genes

After inoculating these three different cell lines with coating for 3 days, total RNA was extracted using a total RNA extraction kit (15596–026, Invitrogen), and the expression of various genes was detected by RT-PCR using synthesized primers (BioTNT, Shanghai, China) for GDNF, BDNF, PMP22, MPZ, CREB, GSK3-β, BMP-2, and VEGF. Their sequences are shown in Supplementary Table 2. In addition, protein expression levels of CREB in SCs, BMP-2, and GSK3-β in MC3T3-E1 and VEGF in HUVECs were measured using Western blotting assays. After extracting the total protein, 30 μg of protein samples in each group were subjected to SDS-PAGE and transferred to the polyvinylidene fluoride (PVDF) membranes. The following primary antibodies were used in the Western blotting assay: anti-CREB antibody (1:1000, Abcam), anti-BMP-2 antibody (1:1000, Abcam), anti-GSK-3β antibody (1:1000, Abcam), anti-VEGF antibody (1:1000, Abcam), and anti-GAPDH antibody (1:5000, YM3029, Immunoway). The PVDF membrane was then probed with a goat antirabbit secondary antibody (1:10000, RS0002, Immunoway). Placed it in a gel imaging system for imaging (Chemi Doc XRS System). The gray value of the protein bands was analyzed by using ImageJ software. All antibodies used in this study are listed in Supporting Information Table 3.

4.5. Influence of SC-Conditioned Medium on MC3T3-E1 Cells

In order to investigate the ability of SCs to regulate osteogenesis on the material surface, SCs were cultured on each group of materials for 3 days. Next, the supernatant was extracted and collected in 1.5 mL centrifuge tubes and centrifuged at 12,000 rpm for 10 min at 4 °C. The collected supernatant was filtered through a 0.22 μm filter and then mixed with material extract in a 1:1 ratio to prepare the conditioned medium, which was later used for coculturing with MC3T3-E1 cells.56−58

MC3T3-E1 cells were first mineralized using a medium containing 10 mM beta glycerin sodium phosphate, 50 mg/mL ascorbic acid-C, and 100 nM of dexamethasone. The medium was then replaced with the previously prepared conditioned medium, and the cells were cultured for seven more days. The supernatant was then collected to assess the ALP activity using a kit (SAE0063, Sigma-Aldrich, USA). After 28 days of induction, ARS was performed for semiquantitative analysis of mineral deposition. Lastly, the cytoskeletal organization of MC3T3-E1 cells from each group was visualized using IF imaging with the operating conditions being consistent with the previous experiment.

4.6. Evaluation of Osteogenesis and Neuroregeneration In Vivo

Animal experiments were approved and carried out in accordance with the guidelines established by the Animal Care and Use Institutional Committee of Lanzhou University (Ethics no. LZUKQ-2021–047). Eight-week-old male specific pathogen-free (SPF) grade Sprague–Dawley rats weighing 350–400 g were provided by the Animal Experiment Center of Lanzhou University. The rats were randomly divided into three groups, namely, the Ti, MAO, and MAO@Li0.05 groups. The implant was long and cylindrical, with a length of 5 mm and a diameter of 1 mm. Rats were anesthetized through an intraperitoneal injection of 10% chloral hydrate (10 wt %, 0.3 mL/100 g) before the implants were placed into their tibiae. The process of implantation is shown in Figure S9A. Four weeks after the operation, the rats were euthanized using an overdose of anesthesia, and the tibiae from both sides were acquired for further analysis. Micro-CT scan was used to quantitatively analyze and detect the bone mass around the implants. In addition, a 3D reconstruction was performed by using the corresponding software (CaseViewer 2.4, 3DHISTECH) to obtain more intuitive visual images and observe the internal microstructure of the samples.

After 4 weeks of implantation, the tibiae were fixed in 4% neutral formaldehyde, decalcified using a 10% EDTA solution for 45 days, and then dehydrated in ethanol and embedded in paraffin to produce for sectioning. The sections were stained using H&E, which facilitated the observation of new bone formation around the implants in each group under different magnifications. At the same time, the main organs (liver, brain, spleen, heart, kidneys, and lungs) of the rats were also stained with H&E to evaluate the biological safety of the implants and to check for any potential side effects in vivo.

These sections were incubated with a mouse monoclonal antineurofilament (NF, 1:1000; Abcam) antibody and a neuropeptide-Y (NPY, 1:250, Proteintech) antibody. Subsequently, they were treated with a biotin-labeled secondary antibody (Histostain-Plus Kits, SP-9001, Zsbio, China) and subjected to DAB staining. The immunohistochemically stained slices were then observed under a microscope (BX53, Olympus, Japan). The quantitative analysis of immunohistochemical staining results was determined using ImageJ software.

4.7. Statistical Analysis

All data were analyzed using SPSS version 27.0 (IBM, Armonk, NY, USA) and are presented as the mean ± the standard deviation (SD). The Shapiro–Wilk test was used to determine whether the data followed a normal distribution. The difference between the groups was analyzed using a one-way analysis of variance and the Kruskal–Wallis H test, with a significance level of *p < 0.05 (*p < 0.05, ** p < 0.01, ***p < 0.001).

Acknowledgments

The work was supported by National Natural Science Foundation of China (82370926, 21904052); National Natural Science Foundation of Gansu Province (23JRRA1080, 20JR10RA641); Fundamental Research Funds for the scientific research fund of School (Hospital) of Stomatology Lanzhou University (lzukqky-2022-t12; lzukqky-2022-t16).

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acsami.4c01520.

  • Flat and cross-sectional SEM images of MAO@Li0.025 and MAO@Li0.075 coatings (Figure S1); quantitative analysis of coatings thickness in each group (Figure S2); AFM topography of the sample surface (Figure S3); roughness statistics for the coatings of each group (Figure S4); XPS high-solution spectra of Ca 2p, P 2p and Ti 2p in the MAO group (Figure S5); Ca and P release and the accumulated amount for MAO, MAO@Li0.025, MAO@Li0.05, and MAO@Li0.075 and friction coefficients and wear depths for Ti, MAO, and MAO@Li0.05 (Figure S6); semiquantitative analysis of SCs nucleus (DAPI) and cytoskeleton (F-actin) staining on the surface of materials in each group (Figure S7); mechanism of Li ions-induced activation of the Wnt/GSK-3β/β-catenin pathway (Figure S8); the implant implantation process and X-ray results of in vivo experiments (Figure S9); H&E-stained images of major organs (Figure S10); atomic concentrations of major elements of MAO and MAO@Li0.075 (Table S1); the sequence for primer target genes used in the study (Table S2); antibodies used for Western blot in this work (Table S3) (PDF)

Author Contributions

○ Q.Z. and S.G. contributed equally to this paper. B.Z., Q.Z., and S.G. contributed to conceptualization. B.L. and Q.L. contributed to formal analysis. K.Z. and J.L. contributed to funding acquisition. Q.Z. and S.G. contributed to methodology. J.H. and B.Z. contributed to project administration. X.L. contributed to software. J.C. and Z.P. contributed to validation. Q.Z. and S.G. contributed to writing—original draft. J.H. and B.Z. contributed to writing—review and editing.

The authors declare no competing financial interest.

Supplementary Material

am4c01520_si_001.pdf (1.3MB, pdf)

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