Reduced graphene oxide loaded with tetrahedral framework nucleic acids for combating orthodontically induced root resorption
Tooth movement (TM) is a mechanics-guided remodeling process in biology, characterized by local aseptic inflammatory response involving the tooth root, alveolar bone, and periodontal ligaments (PDLs) . Currently, with the improvement of the public aesthetic awareness, numerous people undergo orthodontic treatment to improve occlusal function and coordinate facial aesthetics. However, patients are prone to orthodontically induced root resorption (OIRR) during and even after treatment because of the complexity of oral and systemic conditions, the ambiguity of PDLs remodeling, and the inefficiency of TM . As one of the common complications under orthodontic force, OIRR main featured by tooth tissue destruction, second only to enamel leukoplakia. It not only restricts the function of teeth, but also causes the instability of orthodontic effects, thereby severely deteriorates patients’ life quality. Previous studies have shown that, approximately, 90% of patients in orthodontic treatment experience a certain degree of extra-apical root resorption, with the incidence of severe OIRR (exceeding 4 mm) reaching up to 14% . However, owing to the complexity and variability of orthodontic biomechanical systems, high individual differences in PDLs, there is hardly any curative strategy to impede OIRR . Therefore, seeking effective therapy to alleviate or even reverse the pathological changes in OIRR, to achieve efficient, stable, and healthy orthodontic treatment, urgently need to be explored. Recently, a remarkable breakthrough has been made in the advance of nanoscale microstructure from two-dimension (2D) to three-dimension (3D). The research of new nanostructured biomaterials in various diseases has also attracted much attention . Typically, tetrahedral framework nucleic acids (tFNAs) are known to be strategically positioned for its low cytotoxicity, biocompatibility, and membrane penetration in the advancement of cutting-edge fields such as immunotherapy and nano-regenerative medicine . Growing evidence suggests highly complex bone remodeling functions as an indispensable role in the regulation of root resorption . Conversely, dynamic changes in bone are inextricably accompanied with root resorption . Bsp −/− mice exhibit impaired bone healing and extensive tooth root resorption. Skeletal defects observed in Bsp −/− mice was associated with a lack of acellular cementum, which played a central role in the periodontal breakdown and exacerbated pathological changes in periodontal tissue . Root resorption caused by heavy force with severe alveolar bone resorption was related to the increased level of Th17 cells and IL-6 closely . Notably, the tFNA/Pue complex (TPC) restored osteogenesis dysfunction and attenuated the apoptosis of BMSCs in high glucocorticoid microenvironment via the Akt/Bcl-2 and hedgehog pathways . Furthermore, some studies reported that tFNAs facilitated the proliferation of odontogenic stem cells such as dental pulp stem cells , possessed anti-inflammatory and anti-oxidation properties via the inhibition of MAPK signaling in periodontitis . Especially, it ameliorated alveolar bone absorption . Advances have been made to apply nanoscale tFNAs to bone regeneration, however, there are few reports on whether tFNAs affect OIRR. To date, tFNAs have mostly been directly applied to lesions and exert therapeutic effects through diffusion . Nevertheless, the realities of the complex microenvironment around the tooth root, frequent multi-directional mechanical intervention during tooth movement, and continuous updating of gingival crevicular fluid increase the risk of tFNAs being cleared, which becomes a bottleneck for its application. Recently, the combination of nucleic acids with nanomaterials, such as graphene-based nanomaterials, has been substantially advanced over, achieving astonishing functionality, thereby showcasing attractive potential applications . Zhang et al. constructed a highly sequence specific biosensor based on nanocomposites, which are used to detect the specific nucleic acid sequences of SARS-CoV-2 in graphene oxide nanosheets via aggregation-induced emission luminogens (AIEgen)‐labeled oligonucleotide probes . As 2D nanomaterials composed of carbon atom SP2 hybrid orbitals, graphene oxide (GO) shows increasingly broad application prospects in diversified domains of biomedicine . Reduced graphene oxide (rGO) is an important nano-sized derivative of GO. Compared to the GO, the surface of rGO has fewer oxygen-containing functional groups, resulting in a significant improvement in solubility and making the surface exceptionally lively . Previous studies have shown that rGO is easy to functionalize or modify . On the one hand, rGO itself serves as a highly promising biomaterial for disease screening and treatment , on the other hand, it can easily accommodate bioactive molecules such as nucleic acid fragments to improve the delivery efficiency . Hao fabricated a gelatin-alginate rGO hydrogel combined with platelet-derived extracellular vesicles and investigated the possibility of it as a diabetic wound dressing. It modulated immune responses and angiogenesis in the wounds of diabetic rats, and accelerated healing of chronic wounds . Although extensive studies have begun to clarify the biological regulation and therapeutic mechanism related to rGO, to date, whether the rich functional groups of nano-sized rGO can carry tFNAs to reduce their risk of early clearance, achieve sustained release of tFNAs, and ultimately improve OIRR, remain to be elucidated. During the process of mechanical force driving root resorption, there is accompanied by the appearance of periapical bone remodeling, which relies on the organized and orderly coordination of various cells in PDL, such as periodontal ligament cells (PDLCs), osteoclasts, and immune cells . PDLCs have been identified as mesenchymal stem cell reservoir for superior potential to differentiate into osteoblasts in diverse physiological and pathological activities around the roots, including OIRR . Besides, as a mechanical sensitivity effector cell, it directly senses mechanical stimuli in the compressed PDL. Growing evidence indicated that PDLCs facilitated the process of OIRR through producing IL-1β to maintain inflammatory microenvironment and to upregulate osteoclastic activity . However, to date, little is known regarding whether PDLCs is involved in rGO delivery complexes carrying tFNAs to alleviate OIRR. Meanwhile, there is often a negative damage caused by phagocytic cells cleaning of local necrotic tissue during the continuous root absorption process. Macrophages are also mechanically sensitive immune cells, and it has been suggested that their status is a vital link in the occurrence of OIRR . Under force stimulation, the activation of NLRP3 inflammasomes in M1 macrophages by PDLCs would aggravate root resorption . He D revealed that the balance of macrophages affected OIRR. Further studies have shown that the CXCL12/CXCR4 signaling axis mediated OIRR via the upregulation of M1/M2 ratio in PDL . A multifunctional tFNAs system enhanced the crosstalk between schwann cells (SCs) and macrophages, amplified the ability of SCs to recruit macrophages, and facilitated the formation of pro-healing M2 phenotype for nerve regeneration . In Wang’ study, they developed a pioneering DNA nano-drug (TSOs) with the knockdown the expression of SPAK and OSR1. It effectively inhibited the switch of macrophages from M0 to M1 phenotypic and prevented the onset of hydrocephalus . So far, the functions of macrophages in the improvement of OIRR by tFNAs-rGO delivery complex and its specific molecular regulation are poorly understood. Thus, in this study, to clarify whether and how the nanoscale tFNAs-rGO delivery complex affects the root resorption, we used an OIRR model to explore the dynamic changes of root morphology and periapical tissue, osteoclastic activity, and macrophages phenotype in PDLs. Further we established a microenvironment for differentiation induction or polarization induction in vitro to further validate the osteogenesis-enhancement of PDLCs and the polarization-facilitation of M2 phenotype macrophages in ameliorating OIRR mechanistically. Our findings provide further insight into PDLCs and macrophages cell populations in the reconstruction regulation of roots and periodontium and offer a promising strategy for dual-functional nano tFNAs-rGO delivery complex in root resorption therapy. 

Synthesis and characterization of tetrahedral framework nucleic acids (tFNAs) The synthesis of tFNAs was performed as the described in previous studies . The four well-defined DNA nucleic acids single strands (S1, S2, S3, S4) (1 OD DNA) in four tubes were centrifugated, deposited at the tube bottom and then dissolved. The four DNA nucleic acids strands were mixed with equimolar quantities (500 nM, 2 µL) into TM buffer (10 mmol/L Tris-HCl, 50 mmol/LMgCl 2 , pH = 8.0), tFNAs were synthesized by complementary pairing, which were denatured at 95 °C for 10 min and rapidly lowered to 4 °C for 20 min. All DNA nucleic acids sequences are listed in Table . The successful formations of tFNAs were identified by agarose gel electrophoresis. The surface properties of tFNAs were characterized by atomic force microscope (AFM) using an SPM-9700 instrument (Shimadzu, Kyoto, Japan). Transmission electron microscopy (TEM) was used to observe the morphological structure of tFNAs. Additionally, the zeta potential of the sample was evaluated using by dynamic light scattering (DLS) (Nano ZS, Malvern, England). Synthesis of reduced graphene oxide (rGO) and rGO loaded with tFNAs (tFNAs-rGO) According to Kang’s research, the reduction of GO into rGO using vitamin C (VC) as reducing agent . Briefly, graphene oxide (GO) nanosheet (XFNANO, China) was uniformly dispersed in distilled water under ultrasonic action. And 10 mg VC were mixed into 10 mL GO (0.1 mg/mL) nanosheet dispersed suspension. The reduction process was carried out at 95 °C. Removed residual impurities by rinsing, vacuum-dried at 60 °C and collected the rGO. Using self-assembly technology, 100 µL tFNAs (500 nM) were mixed with 100 µL rGO (0.1 mg/mL) in phosphate buffered saline (PBS) and reacted on a fully automatic flip frame at 4 ℃ for 48 h to synthesize tFNAs-rGO delivery complex. Scanning electron microscope (SEM) was used to observe the morphology characteristics of rGO or tFNAs-rGO. Zeta potential was evaluated by DLS. Element composition, molecular properties and crystal structure were analyzed by X-ray photoelectron spectroscopy (XPS, Thermo Kalpha) and X-ray diffraction (XRD, X’Pert PRO MPD). CCK-8 cell viability assay Periodontal ligament cells were isolated from human periodontal membrane tissue as described previously . The cells were routinely cultured in α-MEM medium (Gibco) supplemented with 10% fetal bovine serum (FBS, BioInd, Kibbutz, Israel) and 1% penicillin-streptomycin under 37 °C in a humidified incubator with 5% CO 2 and 95% humidity. When the cells grew to 70% confluence, the cells were passaged by a 25% trypsin with EDTA (Gibco). PDLCs at passage 3–6 were used. RAW264.7 (Shanghai Institutes for Biological Sciences of the Chinese Academy of Sciences, China) were routinely cultured in high glucose DMEM medium (Gibco) containing 10% FBS and 1% penicillin-streptomycin at 37 °C in a humidified incubator with 5% CO 2 and 95% humidity. PDLCs and RAW264.7 were plated in 96-well plates at 3500 cells per well, cultured with medium containing 1 nM, 10 nM, 50 nM, 100 nM rGO or tFNAs-rGO for 48 h. After the treatments, the PDLCs and RAW264.7 incubated in 100 µL of culture medium supplemented with 10 µL of CCK-8 reagent (MCE) at 37 °C for 2.5 h to tests the cell viability. The absorbance was measured at 450 nm using the microplate reader (Synergy H1; BioTek). Animal experiments All procedures were approved by the ethics committee, Fujian Medical University under document number IACUC FJMU 2024-Y-0609. C57BL/6 mice (8 weeks old, male, weighing an average of 20 g) were purchased from Wushi Experimental Animal Limited Company (Fuzhou, China). All the animals were bred and maintained under specific-pathogen-free (SPF) conditions on a 12-/12-h light/dark cycle, with free access to food and water. Animals were anaesthetized with pentobarbital sodium (100 mg/kg, injected intraperitoneally). According to the previous research , 40 g force was applied in orthodontically induced root resorption (OIRR) model induced by the excessive stress. One end of a nickel-titanium coil spring (wire size, 0.2 mm, diameter, 1 mm, Smart Technology) was ligated in the neck of the maxillary first molar with a 0.1 mm stainless steel, and the other end was ligated in the incisor neck (Fig. ). Approximately, the nickel-titanium coil spring provides 40 g of tension force between the maxillary right first molar and incisors. Soft food was feed for 2 days after the operation, and the loading devices were checked daily. If the devices have fallen, they were promptly reloaded. For experiments assessing the effect of the rGO or tFNAs-rGO on OIRR, animals were randomly allocated to four groups: Sham, OIRR, OIRR + rGO, and OIRR + tFNAs-rGO. After local cleaning and drying, periodontal ligament injections were administered to the maxillary first molar using a microinjector . Mice in the rGO or tFNAs-rGO group received periodontal ligament injection of 20 µL rGO or tFNAs-rGO dissolved in PBS every other day. Animals in the Sham and OIRR were treated with equal volume PBS. Animals were sacrificed according to the schematic diagram after different interventions for analyses (Fig. A). Morphometric analysis Animals were sacrificed by anesthetic overdose, the maxillae were separated, fixed by 4% paraformaldehyde solution for 48 h and stored in 70% ethanol at 4 °C before being processed. The morphology of the maxillary molars and the distance of tooth movement were measured under a stereomicroscope (Nikon, SMZ18). Micro-computed tomography Micro-CT scanning of the maxillae of mice was performed by µCT 80 system (filter Al 0.2 mm, 70 kV, 114 µA, SCANCO Medical AG, Switzerland) with the resolution of 5 μm to detect the change of alveolar bone and tooth root. Three-dimensional (3D) images were reconstructed by Mimics and Geomagic Wrap 2017 for morphological assessment. Root resorption areas was represented by red. The imaging system of Micro-CT built-in software was applied for the analysis of resorption lacunae. According to the control side, resorption areas were filled for the analyses of resorption volume. Histology and immunofluorescence staining The maxillae of mice were decalcified using 0.5 M EDTA for 3 weeks, dehydrated, embedded in paraffin, and sectioned at 5 μm used for staining. According to the manufacturer recommendations, the sections were stained with hematoxylin-eosin staining (HE) or tartrate-resistant acid phosphate staining (TRAP). TRAP + cells against root surface with three or more nuclei/cell were identified as odontoclast cells. Observed the absorption of roots and alveolar bone, as well as the osteoclast activity of root surface under the microscope. For immunofluorescence analysis, after antigen retrieval and blocking, frozen sections and fixed RAW264.7 were incubated at 4 °C overnight with primary antibodies against CD86 (ABclonal, 1:200), iNOS (ABclonal, 1:200), CD206 (ABclonal, 1:200), and CD68 (Santa Cruz, 1:50). The secondary antibodies included donkey anti-mouse Alexa Fluor 488/555/647 and donkey anti-rabbit Alexa Fluor 488/555/647 (all from Thermo Fisher Scientific). The nucleus was counterstained with DAPI (Sigma, D9542), the images of the samples were observed by confocal microscope and analyzed with ImageJ software. Sections were blinded and scored by two experienced researchers, and the average scores were used in statistical analyses. Alkaline phosphatase staining PDLCs were seeded in 6-well plates in osteogenesis-inducing medium with rGO or tFNAs-rGO. After 7 and 14 days of osteogenesis-induction, the cells were fixed. According to the instructions, ALP staining was performed (Beyotime Biotechnology), and stained plates were photographed and inspected with a light microscope. Alizarin red staining PDLCs were seeded in 6-well plates in osteogenesis-inducing medium with rGO or tFNAs-rGO. After 21 days of osteogenesis-induction, the cells were fixed. According to the instructions, alizarin red staining was performed (Beyotime Biotechnology), and stained plates were photographed and inspected with a light microscope. Quantitative real-time polymerase chain reaction (qRT-PCR) After the treatments, total RNA was extracted with Trizol (Takara, Tokyo, Japan). The first-strand cDNA was prepared using HiScript II Q RT SuperMix for qPCR (Vazyme Biotech co., China). qRT-PCR was performed using SYBR Premix Ex Taq II (Vazyme Biotech co., China) in CFX96 Real-Time System (Bio-Rad). GAPDH RNA was used as a housekeeping control. The primer sequences are listed in Table . The relative levels of genes expression were normalized to GAPDH and calculated using the 2 −ΔΔCt . Western blotting assay The PDLCs or RAW264.7 lysates were extracted using RIPA buffer for 30 min (4℃). (Beyotime, P0013B) containing phenylmethanesulfonyl fluoride (PMSF, Beyotime, ST505). The concentrations of the Proteins were detected by BCA protein assay kit (Beyotime, #P0012). The samples were heated at 100 °C for 5 min in sample buffer, separated on 10% SDS-polyacrylamide gels, and transferred to PVDF membranes (Bio-Rad). The membranes were blotted with 5% BSA and incubated with primary antibody at 4 °C overnight. The membranes were washed in TBST solution and incubated with the HRP-conjugated secondary antibodies. The antibody-antigen complexes were visualized with ECL reagents (Millipore, WBKLS0100). The following primary antibodies were applied: ALP (Cell Signaling Technology, 1:1000), BSP (Cell Signaling Technology, 1:1000), RUNX-2 (Cell Signaling Technology, 1:1000), OCN (Cell Signaling Technology, 1:1000), COL-1 (Cell Signaling Technology, 1:1000), CD86 (ABclonal, 1:1000), iNOS (ABclonal, 1:1000), CD206 (ABclonal, 1:1000), and GAPDH (ABclonal, 1:5000). Migration assay The effect of rGO or tFNAs-rGO on the migration of RAW264.7 was evaluated using transwell chambers with 8.0 μm pore size (Corning, USA). The lower chamber contained 500 µL of DMEM containing 0.1% FBS with rGO or tFNAs-rGO, and the upper chamber contained RAW264.7 in 200 µL of DMEM containing 0.1% FBS. The chambers were incubated for 48 h (37℃). The RAW264.7 that migrated to the lower chamber were fixed, stained with crystal violet, and counted in three randomly selected microscopic fields per chamber in a blinded manner. Statistical analysis Data are presented as the mean ± standard error of at least three independent experiments. Statistically significant differences were evaluated using two-tailed Student’s t tests for comparison between two groups or by one-way analysis of variance followed by the Tukey’s test for multiple comparisons. All statistical analyses were conducted using GraphPad Prism 8. P < 0.05 was considered statistically significant. 

The synthesis of tFNAs was performed as the described in previous studies . The four well-defined DNA nucleic acids single strands (S1, S2, S3, S4) (1 OD DNA) in four tubes were centrifugated, deposited at the tube bottom and then dissolved. The four DNA nucleic acids strands were mixed with equimolar quantities (500 nM, 2 µL) into TM buffer (10 mmol/L Tris-HCl, 50 mmol/LMgCl 2 , pH = 8.0), tFNAs were synthesized by complementary pairing, which were denatured at 95 °C for 10 min and rapidly lowered to 4 °C for 20 min. All DNA nucleic acids sequences are listed in Table . The successful formations of tFNAs were identified by agarose gel electrophoresis. The surface properties of tFNAs were characterized by atomic force microscope (AFM) using an SPM-9700 instrument (Shimadzu, Kyoto, Japan). Transmission electron microscopy (TEM) was used to observe the morphological structure of tFNAs. Additionally, the zeta potential of the sample was evaluated using by dynamic light scattering (DLS) (Nano ZS, Malvern, England). 

According to Kang’s research, the reduction of GO into rGO using vitamin C (VC) as reducing agent . Briefly, graphene oxide (GO) nanosheet (XFNANO, China) was uniformly dispersed in distilled water under ultrasonic action. And 10 mg VC were mixed into 10 mL GO (0.1 mg/mL) nanosheet dispersed suspension. The reduction process was carried out at 95 °C. Removed residual impurities by rinsing, vacuum-dried at 60 °C and collected the rGO. Using self-assembly technology, 100 µL tFNAs (500 nM) were mixed with 100 µL rGO (0.1 mg/mL) in phosphate buffered saline (PBS) and reacted on a fully automatic flip frame at 4 ℃ for 48 h to synthesize tFNAs-rGO delivery complex. Scanning electron microscope (SEM) was used to observe the morphology characteristics of rGO or tFNAs-rGO. Zeta potential was evaluated by DLS. Element composition, molecular properties and crystal structure were analyzed by X-ray photoelectron spectroscopy (XPS, Thermo Kalpha) and X-ray diffraction (XRD, X’Pert PRO MPD). 

Periodontal ligament cells were isolated from human periodontal membrane tissue as described previously . The cells were routinely cultured in α-MEM medium (Gibco) supplemented with 10% fetal bovine serum (FBS, BioInd, Kibbutz, Israel) and 1% penicillin-streptomycin under 37 °C in a humidified incubator with 5% CO 2 and 95% humidity. When the cells grew to 70% confluence, the cells were passaged by a 25% trypsin with EDTA (Gibco). PDLCs at passage 3–6 were used. RAW264.7 (Shanghai Institutes for Biological Sciences of the Chinese Academy of Sciences, China) were routinely cultured in high glucose DMEM medium (Gibco) containing 10% FBS and 1% penicillin-streptomycin at 37 °C in a humidified incubator with 5% CO 2 and 95% humidity. PDLCs and RAW264.7 were plated in 96-well plates at 3500 cells per well, cultured with medium containing 1 nM, 10 nM, 50 nM, 100 nM rGO or tFNAs-rGO for 48 h. After the treatments, the PDLCs and RAW264.7 incubated in 100 µL of culture medium supplemented with 10 µL of CCK-8 reagent (MCE) at 37 °C for 2.5 h to tests the cell viability. The absorbance was measured at 450 nm using the microplate reader (Synergy H1; BioTek). 

All procedures were approved by the ethics committee, Fujian Medical University under document number IACUC FJMU 2024-Y-0609. C57BL/6 mice (8 weeks old, male, weighing an average of 20 g) were purchased from Wushi Experimental Animal Limited Company (Fuzhou, China). All the animals were bred and maintained under specific-pathogen-free (SPF) conditions on a 12-/12-h light/dark cycle, with free access to food and water. Animals were anaesthetized with pentobarbital sodium (100 mg/kg, injected intraperitoneally). According to the previous research , 40 g force was applied in orthodontically induced root resorption (OIRR) model induced by the excessive stress. One end of a nickel-titanium coil spring (wire size, 0.2 mm, diameter, 1 mm, Smart Technology) was ligated in the neck of the maxillary first molar with a 0.1 mm stainless steel, and the other end was ligated in the incisor neck (Fig. ). Approximately, the nickel-titanium coil spring provides 40 g of tension force between the maxillary right first molar and incisors. Soft food was feed for 2 days after the operation, and the loading devices were checked daily. If the devices have fallen, they were promptly reloaded. For experiments assessing the effect of the rGO or tFNAs-rGO on OIRR, animals were randomly allocated to four groups: Sham, OIRR, OIRR + rGO, and OIRR + tFNAs-rGO. After local cleaning and drying, periodontal ligament injections were administered to the maxillary first molar using a microinjector . Mice in the rGO or tFNAs-rGO group received periodontal ligament injection of 20 µL rGO or tFNAs-rGO dissolved in PBS every other day. Animals in the Sham and OIRR were treated with equal volume PBS. Animals were sacrificed according to the schematic diagram after different interventions for analyses (Fig. A). 

Animals were sacrificed by anesthetic overdose, the maxillae were separated, fixed by 4% paraformaldehyde solution for 48 h and stored in 70% ethanol at 4 °C before being processed. The morphology of the maxillary molars and the distance of tooth movement were measured under a stereomicroscope (Nikon, SMZ18). 

Micro-CT scanning of the maxillae of mice was performed by µCT 80 system (filter Al 0.2 mm, 70 kV, 114 µA, SCANCO Medical AG, Switzerland) with the resolution of 5 μm to detect the change of alveolar bone and tooth root. Three-dimensional (3D) images were reconstructed by Mimics and Geomagic Wrap 2017 for morphological assessment. Root resorption areas was represented by red. The imaging system of Micro-CT built-in software was applied for the analysis of resorption lacunae. According to the control side, resorption areas were filled for the analyses of resorption volume. 

The maxillae of mice were decalcified using 0.5 M EDTA for 3 weeks, dehydrated, embedded in paraffin, and sectioned at 5 μm used for staining. According to the manufacturer recommendations, the sections were stained with hematoxylin-eosin staining (HE) or tartrate-resistant acid phosphate staining (TRAP). TRAP + cells against root surface with three or more nuclei/cell were identified as odontoclast cells. Observed the absorption of roots and alveolar bone, as well as the osteoclast activity of root surface under the microscope. For immunofluorescence analysis, after antigen retrieval and blocking, frozen sections and fixed RAW264.7 were incubated at 4 °C overnight with primary antibodies against CD86 (ABclonal, 1:200), iNOS (ABclonal, 1:200), CD206 (ABclonal, 1:200), and CD68 (Santa Cruz, 1:50). The secondary antibodies included donkey anti-mouse Alexa Fluor 488/555/647 and donkey anti-rabbit Alexa Fluor 488/555/647 (all from Thermo Fisher Scientific). The nucleus was counterstained with DAPI (Sigma, D9542), the images of the samples were observed by confocal microscope and analyzed with ImageJ software. Sections were blinded and scored by two experienced researchers, and the average scores were used in statistical analyses. 

PDLCs were seeded in 6-well plates in osteogenesis-inducing medium with rGO or tFNAs-rGO. After 7 and 14 days of osteogenesis-induction, the cells were fixed. According to the instructions, ALP staining was performed (Beyotime Biotechnology), and stained plates were photographed and inspected with a light microscope. 

PDLCs were seeded in 6-well plates in osteogenesis-inducing medium with rGO or tFNAs-rGO. After 21 days of osteogenesis-induction, the cells were fixed. According to the instructions, alizarin red staining was performed (Beyotime Biotechnology), and stained plates were photographed and inspected with a light microscope. 

After the treatments, total RNA was extracted with Trizol (Takara, Tokyo, Japan). The first-strand cDNA was prepared using HiScript II Q RT SuperMix for qPCR (Vazyme Biotech co., China). qRT-PCR was performed using SYBR Premix Ex Taq II (Vazyme Biotech co., China) in CFX96 Real-Time System (Bio-Rad). GAPDH RNA was used as a housekeeping control. The primer sequences are listed in Table . The relative levels of genes expression were normalized to GAPDH and calculated using the 2 −ΔΔCt . 

The PDLCs or RAW264.7 lysates were extracted using RIPA buffer for 30 min (4℃). (Beyotime, P0013B) containing phenylmethanesulfonyl fluoride (PMSF, Beyotime, ST505). The concentrations of the Proteins were detected by BCA protein assay kit (Beyotime, #P0012). The samples were heated at 100 °C for 5 min in sample buffer, separated on 10% SDS-polyacrylamide gels, and transferred to PVDF membranes (Bio-Rad). The membranes were blotted with 5% BSA and incubated with primary antibody at 4 °C overnight. The membranes were washed in TBST solution and incubated with the HRP-conjugated secondary antibodies. The antibody-antigen complexes were visualized with ECL reagents (Millipore, WBKLS0100). The following primary antibodies were applied: ALP (Cell Signaling Technology, 1:1000), BSP (Cell Signaling Technology, 1:1000), RUNX-2 (Cell Signaling Technology, 1:1000), OCN (Cell Signaling Technology, 1:1000), COL-1 (Cell Signaling Technology, 1:1000), CD86 (ABclonal, 1:1000), iNOS (ABclonal, 1:1000), CD206 (ABclonal, 1:1000), and GAPDH (ABclonal, 1:5000). 

The effect of rGO or tFNAs-rGO on the migration of RAW264.7 was evaluated using transwell chambers with 8.0 μm pore size (Corning, USA). The lower chamber contained 500 µL of DMEM containing 0.1% FBS with rGO or tFNAs-rGO, and the upper chamber contained RAW264.7 in 200 µL of DMEM containing 0.1% FBS. The chambers were incubated for 48 h (37℃). The RAW264.7 that migrated to the lower chamber were fixed, stained with crystal violet, and counted in three randomly selected microscopic fields per chamber in a blinded manner. 

Data are presented as the mean ± standard error of at least three independent experiments. Statistically significant differences were evaluated using two-tailed Student’s t tests for comparison between two groups or by one-way analysis of variance followed by the Tukey’s test for multiple comparisons. All statistical analyses were conducted using GraphPad Prism 8. P < 0.05 was considered statistically significant. 

Preparation, synthesis, and identification of tFNAs and tFNAs-rGO The successful synthesis of nano tFNAs is the foundation for building the delivery complex of reduced graphene oxide (rGO) carrying tetrahedral framework nucleic acids (tFNAs). First, we synthesized the four specifically designed isometric single-strand DNAs (ssDNA) strands and ssDNA strands were easily assembled into stable tFNAs through an annealing process. To prove that we successfully obtained tFNAs, some of the main characteristics of tFNAs, such as the morphology and average potential, were detected. As illustrated by the polyacrylamide gel electrophoresis (PAGE) shown in Fig. A, the result demonstrated the formed S1, S2, S3, S4, and tFNAs, respectively. It was obvious that the migration rate of tFNAs was slower than that other ssDNA strands. Additionally, tFNAs were effectively formed and displayed clear and highlighted band. The morphology of tFNAs were characterized by atomic force microscopy (AFM) and transmission electron microscopy (TEM). The nanoparticle-like framework morphology of tFNAs was about 10–20 nm in a dry state (Fig. B). As we observed from TEM imaging, the shape of the tFNAs could be recognized, manifesting circular or approximately triangular particles with well-dispersed phenomenon (Fig. C). The results aligned with that of previous studies . Dynamic light scattering (DLS) tests were conducted to explore the zeta potential of tFNAs. Similarly, Fig. D showed tFNAs were negatively charged and the average zeta potential was approximately − 1.49 mV. Altogether, the results indicated that tFNAs were efficiently and successfully assembled. Second, we employed self-assembly technology to attach reduced graphene oxide (rGO) with the synthesized tFNAs to build the delivery complex under co-incubation at 4 °C. The morphology feature of nano sized tFNAs-rGO was analyzed by scanning electron microscope (SEM) (Fig. A). The outcomes revealed that rGO appeared as layered flakes, possessed flowing and smooth surface with a thickness of approximately 1–3 nm. It showed that tFNAs nanoparticles were evenly distributed the surface of the rGO flaky-like structure in delivery complex. Subsequent analyses using DLS were performed to explore the zeta potential of rGO and tFNAs-rGO. As shown in Fig. B, both were negatively charged, with values of -3.62 mV and − 2.21 mV, respectively. To further verify the successful synthesis of tFNAs-rGO, X-ray photoelectron spectroscopy (XPS) was carried out to detect the composition, chemical and electronic states of various elements in rGO or tFNAs-rGO (Fig. C). XPS analysis indicated the differences in chemical composition of rGO and tFNAs-rGO. Full spectra and peaks fitting of the samples are shown in Fig. C. Compared with the rGO group, there was C-N bonds in the tFNAs-rGO group (Fig. C-a and C-b). Meanwhile, P-O bonds and C = N were also detected in the tFNAs-rGO group (Fig. C-c and C-d). Besides, according to atomic quantitative analysis results, the proportion of phosphorus element in the tFNAs-rGO group (3.35%) was higher than that in the rGO group (0.71%), partly suggesting the effective introduction of tFNAs. The loading of tFNAs on the surface rGO nanosheets were confirmed by powder XRD and are in Fig. D. The diffraction pattern of rGO centered at 28.68° (0 0 2). In addition to the peak of rGO, another peak appeared at 42.5°, which indicated that tFNAs successfully loaded onto rGO nanosheets during the self-assembly process. In addition, CCK-8 experiments showed that rGO and tFNAs-rGO had no effects on the proliferation or survival of PDLCs and RAW264.7 cells at concentrations ranged from 1 to 100nM (Fig. E). It indicated that the tFNAs-rGO delivery complex had biosafety and low toxicity. Taken together, these findings indicated that tFNAs was loaded into rGO and we successfully built nanoscale tFNAs-rGO delivery complex. tFNAs-rGO alleviates OIRR and downregulates periapical osteoclast activity To explore the effect of tFNAs-rGO delivery complex on mechanical force induced root resorption (Fig. A), we performed 3D reconstruction of the tooth roots using Micro-CT and examined the dynamic changes of the root resorption area upon force application and after applying tFNAs-rGO. The Micro-CT analysis on the 14th day showed that remarkable root resorption appeared after force application, which was consistent with previous research . However, after the treatment of rGO and tFNAs-rGO, root resorption volume decreased significantly (Fig. B, S2). Notably, compared with rGO, tFNAs-rGO acted enhanced therapeutic efficacy in OIRR. Moreover, we applied HE staining to evaluate the area of root resorption at the histological level. Consistently, as Fig. C shows, the root absorption area in the OIRR group was prominently increased. However, the level of root absorption in the group exposure to rGO or tFNAs-rGO was sharply abolished compared with that of the OIRR group. As expected, the root absorption area in the tFNAs-rGO group was lower than that in the rGO group. The osteoclast is the central player of bone formation and resorption. The knowledge of regulation mechanisms of osteoclast activity is the basis for understanding the whole huge and complex network for root-bone remodeling . Further, the osteoclastogenic activity around the tooth roots was measured using TRAP staining. As seen in Fig. D, the osteoclast activity markedly upregulated and the number of osteoclasts gradually increased in the periapical tissue of OIRR mice. Nevertheless, rGO and tFNAs-rGO abrogated the OIRR-induced enhancement of osteoclast activity. And whether on Day 7 or Day 14, the tFNAs-rGO group had the lowest number of osteoclasts among the three groups of modeling. Collectively, these findings suggested that tFNAs-rGO delivery complex relieved root resorption and downregulated the level of periapical osteoclast activity. tFNAs-rGO boosts the ability of osteogenesis in PDLCs To further elucidate the precise mechanism by which tFNAs-rGO delivery complex ameliorates OIRR, we first examined the effect of tFNAs-rGO on the osteogenic differentiation ability of PDLCs. On the one hand, the effects of tFNAs-rGO on the osteogenic differentiation ability of PDLCs were assessed through morphological staining. The ALP staining results showed that the obvious blue precipitates appeared in the cytoplasm of PDLCs treated with rGO and tFNAs-rGO. Besides, among the three groups, the number of positive blue stained PDLCs was the highest in the tFNAs-rGO group (Fig. A). In line with the ALP staining data, alizarin red staining assays also showed that, after 21 days of osteogenic induction, both the rGO group and the tFNAs-rGO group showed significant dark red compounds compared to the Con group. Consistently, there were more red mineralized nodules with deeper staining in the tFNAs-rGO group compared to that in the rGO group (Fig. B). On the other hand, we validated the enhancement of PDLCs osteogenic differentiation by tFNAs-rGO at the molecular level using quantitative real-time PCR (qRT-PCR) and western blot. As shown in Fig. C, after tFNAs-rGO was introduced into PDLCs, the mRNA level of osteogenesis related genes, such as Ocn , Opn , Osterix , and Runx-2 , elevated sharply. And Alp mRNA level was significantly higher in the tFNAs-rGO group than that in the other two groups at 21 days ( P < 0.05 ). Moreover, compared to the rGO group, the tFNAs-rGO group showed a more significant improvement on the expressions of osteogenic genes. According to results from the western blot, tFNAs-rGO led to remarkable induction of osteogenic differentiation, as the protein expressions of the ALP, BSP, RUNX-2, and OCN were notably enhanced following the addition of tFNAs-rGO. Furthermore, compared to that in the rGO groups, the protein level of ALP, BSP, RUNX-2, and OCN were markedly upregulated in the tFNAs-rGO group (Fig. D). These data demonstrated that tFNAs-rGO boosted the ability of osteogenesis in PDLCs. tFNAs-rGO reverses OIRR-enhanced M1/M2 macrophage ratio in mice Existing studies have shown that macrophages are also implicated as important effector cells in OIRR . Mechanical force triggered M1/M2 macrophage ratio in the PDL, which participated in the occurrence and progress of OIRR . To further clarify whether the tFNAs-rGO delivery complex had an impact on the biological status of macrophages, we performed immunofluorescence staning to detect and quantify the changes of macrophage polarization status in the periodontal ligament of mice after intervention. As shown in Fig. A and B, CD68 + iNOS + macrophages drastically accumulated, coupled with a decrease in the number of CD68 + CD163 + macrophages, after force application. However, these changes were blunted by in situ injection of rGO or tFNAs-rGO (Fig. A, B). Consequently, the results implied that the number of M1 pro-inflammatory macrophages was upregulated in the periodontal ligament of mice, while M2 anti-inflammatory macrophages reduced during the mechanically induced root resorption. It is worth noting that after the treatment of rGO or tFNAs-rGO, the number of CD68 + iNOS + M1 macrophages decreased. Meanwhile, the number of CD68 + CD163 + M2 macrophages in the rGO or tFNAs-rGO group was elevated significantly compared to OIRR mice during force application. Expectedly, it showed that the blockage of M1 macrophages accumulation and the promotion on M2 macrophage recruitment were more distinct in the tFNAs-rGO group compared with the rGO group (Fig. A, B). Next, we quantitatively analyzed the proportion of M1 and M2 macrophages in the periodontal ligament. OIRR led to a pronounced increase in the ratio of M1/M2 macrophages. But the treatment of tFNAs-rGO reversed the OIRR-triggered rise of the M1/M macrophage ratio (Fig. C). These results revealed part of the mechanism by which tFNAs-rGO alleviated OIRR by reversing OIRR-enhanced M1/M2 macrophage ratio in mice. tFNAs-rGO suppresses M1 and promotes M2 polarization in vitro To further clarify the mechanism by which tFNAs-rGO delivery complex affected the polarization state of macrophages, we assessed the expression of M1 and M2 markers in RAW264.7-derived macrophages after rGO or tFNAs-rGO stimulation using WB and qRT-PCR analysis in vitro. Specifically, in line with the in vivo data, in vitro assays also showed that RAW264.7 stimulated with LPS differentiated into M1-like pro-inflammatory macrophages, as evidenced by increased mRNA levels Tnf-α and Inos (Fig. A). However, the raised levels of Tnf-α and Inos were rescued by rGO or tFNAs-rGO application (Fig. A). Additionally, rGO and tFNAs-rGO could still obviously downregulate the expression of Tnf-α , Inos , and Il-1β when LPS was not introduced into the culture system (Fig. A). These results indicated that tFNAs-rGO could effectively reduce the expression of the inflammatory factors in macrophages stimulated by LPS. Compared with the Con group and the IL-4 group, the expression of M2-like anti-inflammatory macrophages related-genes in RAW264.7 cells, such as Arg-1 , Tgf-β , and Cd206 , sharply increased in the IL-4 + tFNAs-rGO group. Besides, there was higher mRNA levels of Tgf-β and Cd206 in RAW264.7 cells of IL-4 + tFNAs-rGO group compared to IL-4 + rGO group (Fig. B). Meanwhile, compared to the Con group, the expression of M2 macrophage polarization related genes, such as Cd206 and Tgf-β but not Arg-1 , strikingly increased in the tFNAs-rGO group (Fig. B). Western blot was used to detect changes in RAW264.7 cells at the protein level. As presented in Fig. D, the upregulated expression of iNOS and CD68 in LPS-treated RAW264.7 cells was markedly reversed after rGO or tFNAs-rGO administration in vitro. Conversely, CD163 and CD206 expression in the tFNAs-rGO group was significantly higher than that in the IL-4 group ( P < 0.05 ). Moreover, it showed a greater promotion in the protein levels of CD163 and CD206, markers of M2 macrophages, in the tFNAs-rGO group compared to the rGO group (Fig. D). To further verify the postulation that tFNAs-rGO prevented M1 macrophages differentiation and induced M2 macrophage polarization, immunofluorescence staining was utilized. Consistently, tFNAs-rGO treatment was observed to decrease the numbers of iNOS + M1 macrophages in the LPS + tFNAs-rGO group significantly compared to that in the LPS group (Fig. E). Most notably, compared with rGO, tFNAs-rGO exhibited a more prominent inhibition on the formation of iNOS + M1 macrophages. Furthermore, tFNAs-rGO boosted the number of CD163 + M2 macrophage induced by IL-4, whereas there was no significant enhancement on the CD163 + M2 macrophage number in the rGO group compared with IL-4 group (Fig. F). The administration of tFNAs-rGO induced a reduction of iNOS + or CD86 + M1 macrophages, but a rise of CD163 + or CD206 + M2 macrophages (Fig. C, D). We performed transwell migration assay to test the chemotactic ability of rGO or tFNAs-rGO to attract macrophages in vitro. Interestingly, tFNAs-rGO or rGO significantly increased the number of RAW264.7 cells in the lower chamber. In addition, the promotion effect on migration ability of RAW264.7 cells was more pronounced in the tFNAs-rGO group compared to the rGO group (Fig. E). Collectively, these findings identified tFNAs-rGO affected macrophage status via the suppression M1 and the promotion M2 polarization for orthodontically induced root resorption remission. tFNAs-rGO shows no obvious organ toxicity and does not affect the tooth movement efficiency in vivo To investigate whether the tFNAs-rGO delivery complex had toxic effects on animals during application (Fig. A, S1), we employed HE staining to evaluate the changes in the cellular morphology and tissue structure of organs such as liver, kidney, and heart. HE staining showed that the structures of liver lobular could be clearly seen in these four groups, with liver cells arranged radially around the central vein. Besides, hepatocytes were intact and had abundant cytoplasm, and the nuclei were circular and located in the center, without significant enlargement (Fig. A). As shown in Fig. B, the size and morphology of the glomerulus are normal with clear boundaries in the OIRR + rGO group and OIRR + tFNAs-rGO group. Similarly, the brush inside the renal tubules is intact without significant expansion after rGO or tFNAs-rGO treatment. There were large amounts of proximal tubules composed of cubic epithelial cells with nuclei located at the base and regular distal tubules. Compared to the Sham group, there was no obvious infiltration of inflammatory cells in the renal interstitium in the OIRR + rGO group and OIRR + tFNAs-rGO group. In all groups, the muscle fibers were slender and cylindrical, connected to each other to form a network in the longitudinal section. The muscle fibers were short cylindrical or polygonal in shape, with similar sizes in the transverse section. There was a small amount of connective tissue and capillaries between the muscle fibers. These data implied that rGO and tFNAs-rGO had no significant organ toxic effects on OIRR mice. To further clarify the effects of rGO and tFNAs-rGO delivery complex on the efficiency of tooth movement induced by orthodontic force, we measured and analyzed the distance of tooth movement in different groups using a stereomicroscope and Micro-CT (Fig. B). Compared to the Sham group, the OIRR group showed a significant increase in tooth movement distance. It is worth noting that the distance of tooth movement did not significantly change with the application of rGO and tFNAs-rGO on the 7th and 14th days. The above data indicated that tFNAs-rGO exhibited no effect on the tooth movement efficiency of OIRR in vivo. 

The successful synthesis of nano tFNAs is the foundation for building the delivery complex of reduced graphene oxide (rGO) carrying tetrahedral framework nucleic acids (tFNAs). First, we synthesized the four specifically designed isometric single-strand DNAs (ssDNA) strands and ssDNA strands were easily assembled into stable tFNAs through an annealing process. To prove that we successfully obtained tFNAs, some of the main characteristics of tFNAs, such as the morphology and average potential, were detected. As illustrated by the polyacrylamide gel electrophoresis (PAGE) shown in Fig. A, the result demonstrated the formed S1, S2, S3, S4, and tFNAs, respectively. It was obvious that the migration rate of tFNAs was slower than that other ssDNA strands. Additionally, tFNAs were effectively formed and displayed clear and highlighted band. The morphology of tFNAs were characterized by atomic force microscopy (AFM) and transmission electron microscopy (TEM). The nanoparticle-like framework morphology of tFNAs was about 10–20 nm in a dry state (Fig. B). As we observed from TEM imaging, the shape of the tFNAs could be recognized, manifesting circular or approximately triangular particles with well-dispersed phenomenon (Fig. C). The results aligned with that of previous studies . Dynamic light scattering (DLS) tests were conducted to explore the zeta potential of tFNAs. Similarly, Fig. D showed tFNAs were negatively charged and the average zeta potential was approximately − 1.49 mV. Altogether, the results indicated that tFNAs were efficiently and successfully assembled. Second, we employed self-assembly technology to attach reduced graphene oxide (rGO) with the synthesized tFNAs to build the delivery complex under co-incubation at 4 °C. The morphology feature of nano sized tFNAs-rGO was analyzed by scanning electron microscope (SEM) (Fig. A). The outcomes revealed that rGO appeared as layered flakes, possessed flowing and smooth surface with a thickness of approximately 1–3 nm. It showed that tFNAs nanoparticles were evenly distributed the surface of the rGO flaky-like structure in delivery complex. Subsequent analyses using DLS were performed to explore the zeta potential of rGO and tFNAs-rGO. As shown in Fig. B, both were negatively charged, with values of -3.62 mV and − 2.21 mV, respectively. To further verify the successful synthesis of tFNAs-rGO, X-ray photoelectron spectroscopy (XPS) was carried out to detect the composition, chemical and electronic states of various elements in rGO or tFNAs-rGO (Fig. C). XPS analysis indicated the differences in chemical composition of rGO and tFNAs-rGO. Full spectra and peaks fitting of the samples are shown in Fig. C. Compared with the rGO group, there was C-N bonds in the tFNAs-rGO group (Fig. C-a and C-b). Meanwhile, P-O bonds and C = N were also detected in the tFNAs-rGO group (Fig. C-c and C-d). Besides, according to atomic quantitative analysis results, the proportion of phosphorus element in the tFNAs-rGO group (3.35%) was higher than that in the rGO group (0.71%), partly suggesting the effective introduction of tFNAs. The loading of tFNAs on the surface rGO nanosheets were confirmed by powder XRD and are in Fig. D. The diffraction pattern of rGO centered at 28.68° (0 0 2). In addition to the peak of rGO, another peak appeared at 42.5°, which indicated that tFNAs successfully loaded onto rGO nanosheets during the self-assembly process. In addition, CCK-8 experiments showed that rGO and tFNAs-rGO had no effects on the proliferation or survival of PDLCs and RAW264.7 cells at concentrations ranged from 1 to 100nM (Fig. E). It indicated that the tFNAs-rGO delivery complex had biosafety and low toxicity. Taken together, these findings indicated that tFNAs was loaded into rGO and we successfully built nanoscale tFNAs-rGO delivery complex. 

To explore the effect of tFNAs-rGO delivery complex on mechanical force induced root resorption (Fig. A), we performed 3D reconstruction of the tooth roots using Micro-CT and examined the dynamic changes of the root resorption area upon force application and after applying tFNAs-rGO. The Micro-CT analysis on the 14th day showed that remarkable root resorption appeared after force application, which was consistent with previous research . However, after the treatment of rGO and tFNAs-rGO, root resorption volume decreased significantly (Fig. B, S2). Notably, compared with rGO, tFNAs-rGO acted enhanced therapeutic efficacy in OIRR. Moreover, we applied HE staining to evaluate the area of root resorption at the histological level. Consistently, as Fig. C shows, the root absorption area in the OIRR group was prominently increased. However, the level of root absorption in the group exposure to rGO or tFNAs-rGO was sharply abolished compared with that of the OIRR group. As expected, the root absorption area in the tFNAs-rGO group was lower than that in the rGO group. The osteoclast is the central player of bone formation and resorption. The knowledge of regulation mechanisms of osteoclast activity is the basis for understanding the whole huge and complex network for root-bone remodeling . Further, the osteoclastogenic activity around the tooth roots was measured using TRAP staining. As seen in Fig. D, the osteoclast activity markedly upregulated and the number of osteoclasts gradually increased in the periapical tissue of OIRR mice. Nevertheless, rGO and tFNAs-rGO abrogated the OIRR-induced enhancement of osteoclast activity. And whether on Day 7 or Day 14, the tFNAs-rGO group had the lowest number of osteoclasts among the three groups of modeling. Collectively, these findings suggested that tFNAs-rGO delivery complex relieved root resorption and downregulated the level of periapical osteoclast activity. 

To further elucidate the precise mechanism by which tFNAs-rGO delivery complex ameliorates OIRR, we first examined the effect of tFNAs-rGO on the osteogenic differentiation ability of PDLCs. On the one hand, the effects of tFNAs-rGO on the osteogenic differentiation ability of PDLCs were assessed through morphological staining. The ALP staining results showed that the obvious blue precipitates appeared in the cytoplasm of PDLCs treated with rGO and tFNAs-rGO. Besides, among the three groups, the number of positive blue stained PDLCs was the highest in the tFNAs-rGO group (Fig. A). In line with the ALP staining data, alizarin red staining assays also showed that, after 21 days of osteogenic induction, both the rGO group and the tFNAs-rGO group showed significant dark red compounds compared to the Con group. Consistently, there were more red mineralized nodules with deeper staining in the tFNAs-rGO group compared to that in the rGO group (Fig. B). On the other hand, we validated the enhancement of PDLCs osteogenic differentiation by tFNAs-rGO at the molecular level using quantitative real-time PCR (qRT-PCR) and western blot. As shown in Fig. C, after tFNAs-rGO was introduced into PDLCs, the mRNA level of osteogenesis related genes, such as Ocn , Opn , Osterix , and Runx-2 , elevated sharply. And Alp mRNA level was significantly higher in the tFNAs-rGO group than that in the other two groups at 21 days ( P < 0.05 ). Moreover, compared to the rGO group, the tFNAs-rGO group showed a more significant improvement on the expressions of osteogenic genes. According to results from the western blot, tFNAs-rGO led to remarkable induction of osteogenic differentiation, as the protein expressions of the ALP, BSP, RUNX-2, and OCN were notably enhanced following the addition of tFNAs-rGO. Furthermore, compared to that in the rGO groups, the protein level of ALP, BSP, RUNX-2, and OCN were markedly upregulated in the tFNAs-rGO group (Fig. D). These data demonstrated that tFNAs-rGO boosted the ability of osteogenesis in PDLCs. 

Existing studies have shown that macrophages are also implicated as important effector cells in OIRR . Mechanical force triggered M1/M2 macrophage ratio in the PDL, which participated in the occurrence and progress of OIRR . To further clarify whether the tFNAs-rGO delivery complex had an impact on the biological status of macrophages, we performed immunofluorescence staning to detect and quantify the changes of macrophage polarization status in the periodontal ligament of mice after intervention. As shown in Fig. A and B, CD68 + iNOS + macrophages drastically accumulated, coupled with a decrease in the number of CD68 + CD163 + macrophages, after force application. However, these changes were blunted by in situ injection of rGO or tFNAs-rGO (Fig. A, B). Consequently, the results implied that the number of M1 pro-inflammatory macrophages was upregulated in the periodontal ligament of mice, while M2 anti-inflammatory macrophages reduced during the mechanically induced root resorption. It is worth noting that after the treatment of rGO or tFNAs-rGO, the number of CD68 + iNOS + M1 macrophages decreased. Meanwhile, the number of CD68 + CD163 + M2 macrophages in the rGO or tFNAs-rGO group was elevated significantly compared to OIRR mice during force application. Expectedly, it showed that the blockage of M1 macrophages accumulation and the promotion on M2 macrophage recruitment were more distinct in the tFNAs-rGO group compared with the rGO group (Fig. A, B). Next, we quantitatively analyzed the proportion of M1 and M2 macrophages in the periodontal ligament. OIRR led to a pronounced increase in the ratio of M1/M2 macrophages. But the treatment of tFNAs-rGO reversed the OIRR-triggered rise of the M1/M macrophage ratio (Fig. C). These results revealed part of the mechanism by which tFNAs-rGO alleviated OIRR by reversing OIRR-enhanced M1/M2 macrophage ratio in mice. 

To further clarify the mechanism by which tFNAs-rGO delivery complex affected the polarization state of macrophages, we assessed the expression of M1 and M2 markers in RAW264.7-derived macrophages after rGO or tFNAs-rGO stimulation using WB and qRT-PCR analysis in vitro. Specifically, in line with the in vivo data, in vitro assays also showed that RAW264.7 stimulated with LPS differentiated into M1-like pro-inflammatory macrophages, as evidenced by increased mRNA levels Tnf-α and Inos (Fig. A). However, the raised levels of Tnf-α and Inos were rescued by rGO or tFNAs-rGO application (Fig. A). Additionally, rGO and tFNAs-rGO could still obviously downregulate the expression of Tnf-α , Inos , and Il-1β when LPS was not introduced into the culture system (Fig. A). These results indicated that tFNAs-rGO could effectively reduce the expression of the inflammatory factors in macrophages stimulated by LPS. Compared with the Con group and the IL-4 group, the expression of M2-like anti-inflammatory macrophages related-genes in RAW264.7 cells, such as Arg-1 , Tgf-β , and Cd206 , sharply increased in the IL-4 + tFNAs-rGO group. Besides, there was higher mRNA levels of Tgf-β and Cd206 in RAW264.7 cells of IL-4 + tFNAs-rGO group compared to IL-4 + rGO group (Fig. B). Meanwhile, compared to the Con group, the expression of M2 macrophage polarization related genes, such as Cd206 and Tgf-β but not Arg-1 , strikingly increased in the tFNAs-rGO group (Fig. B). Western blot was used to detect changes in RAW264.7 cells at the protein level. As presented in Fig. D, the upregulated expression of iNOS and CD68 in LPS-treated RAW264.7 cells was markedly reversed after rGO or tFNAs-rGO administration in vitro. Conversely, CD163 and CD206 expression in the tFNAs-rGO group was significantly higher than that in the IL-4 group ( P < 0.05 ). Moreover, it showed a greater promotion in the protein levels of CD163 and CD206, markers of M2 macrophages, in the tFNAs-rGO group compared to the rGO group (Fig. D). To further verify the postulation that tFNAs-rGO prevented M1 macrophages differentiation and induced M2 macrophage polarization, immunofluorescence staining was utilized. Consistently, tFNAs-rGO treatment was observed to decrease the numbers of iNOS + M1 macrophages in the LPS + tFNAs-rGO group significantly compared to that in the LPS group (Fig. E). Most notably, compared with rGO, tFNAs-rGO exhibited a more prominent inhibition on the formation of iNOS + M1 macrophages. Furthermore, tFNAs-rGO boosted the number of CD163 + M2 macrophage induced by IL-4, whereas there was no significant enhancement on the CD163 + M2 macrophage number in the rGO group compared with IL-4 group (Fig. F). The administration of tFNAs-rGO induced a reduction of iNOS + or CD86 + M1 macrophages, but a rise of CD163 + or CD206 + M2 macrophages (Fig. C, D). We performed transwell migration assay to test the chemotactic ability of rGO or tFNAs-rGO to attract macrophages in vitro. Interestingly, tFNAs-rGO or rGO significantly increased the number of RAW264.7 cells in the lower chamber. In addition, the promotion effect on migration ability of RAW264.7 cells was more pronounced in the tFNAs-rGO group compared to the rGO group (Fig. E). Collectively, these findings identified tFNAs-rGO affected macrophage status via the suppression M1 and the promotion M2 polarization for orthodontically induced root resorption remission. 

To investigate whether the tFNAs-rGO delivery complex had toxic effects on animals during application (Fig. A, S1), we employed HE staining to evaluate the changes in the cellular morphology and tissue structure of organs such as liver, kidney, and heart. HE staining showed that the structures of liver lobular could be clearly seen in these four groups, with liver cells arranged radially around the central vein. Besides, hepatocytes were intact and had abundant cytoplasm, and the nuclei were circular and located in the center, without significant enlargement (Fig. A). As shown in Fig. B, the size and morphology of the glomerulus are normal with clear boundaries in the OIRR + rGO group and OIRR + tFNAs-rGO group. Similarly, the brush inside the renal tubules is intact without significant expansion after rGO or tFNAs-rGO treatment. There were large amounts of proximal tubules composed of cubic epithelial cells with nuclei located at the base and regular distal tubules. Compared to the Sham group, there was no obvious infiltration of inflammatory cells in the renal interstitium in the OIRR + rGO group and OIRR + tFNAs-rGO group. In all groups, the muscle fibers were slender and cylindrical, connected to each other to form a network in the longitudinal section. The muscle fibers were short cylindrical or polygonal in shape, with similar sizes in the transverse section. There was a small amount of connective tissue and capillaries between the muscle fibers. These data implied that rGO and tFNAs-rGO had no significant organ toxic effects on OIRR mice. To further clarify the effects of rGO and tFNAs-rGO delivery complex on the efficiency of tooth movement induced by orthodontic force, we measured and analyzed the distance of tooth movement in different groups using a stereomicroscope and Micro-CT (Fig. B). Compared to the Sham group, the OIRR group showed a significant increase in tooth movement distance. It is worth noting that the distance of tooth movement did not significantly change with the application of rGO and tFNAs-rGO on the 7th and 14th days. The above data indicated that tFNAs-rGO exhibited no effect on the tooth movement efficiency of OIRR in vivo. 

The advantages of nanoscale system in disease therapy lie in their desirable characteristics, including nanoscale size, excellent biocompatibility, targeted applicability, and controlled release and more. By using an in vivo mechanical force-induced root resorption model and in vitro an osteogenic induced microenvironment or cells polarization system, our study uncovers the pivotal roles and the mechanisms of the dual-functional tFNAs-rGO delivery complex at the nanoscale in alleviating orthodontically induced root resorption (OIRR) and PDLCs or macrophages function during root tissue reconstruction (Fig. ). Root resorption is the pathological loss of dental hard tissue and is triggered by odontoclastic action. It may appear on the outer aspect of the root or within the root canal, however, regardless of its region, it is irreversible and may result in a decline in chewing function for patients, and in severe cases, may even cause tooth loss . Clinically, root resorption is a long-lasting issue due to its ambiguity and complexity of etiology. Previous studies demonstrated that canal filling techniques and regenerative endodontic procedures were able to arrest the resorption, reduced the size of the resorptive area and ultimately improved the longevity and restorability of teeth with internal root resorption . However, it is frustrating to note that there are almost no effective treatment strategies for external surface root resorption. The external surface root resorption is usually an adverse outcome of orthodontic tooth movement . It has been reported that the incidence of orthodontically induced root resorption (OIRR) is extremely high in orthodontic patients . To date, only by the management of appropriately eliminating etiological factors, such as suspending orthodontic treatment, can the root absorption be slowed down. Therefore, there is an urgent need to explore new strategies for treating OIRR. In recent years, nanoscale materials have been applied in the diagnosis and treatment of various diseases, including the stomatology . A mitochondrial complex biomimetic nanozyme (MCBN), with biological properties like natural complexes and nanoscale size, demonstrated anti-inflammatory effect and relieved alveolar bone loss by targeting the mitochondrial electron transport chain homeostasis of macrophages to inhibit the activation of NLRP3 inflammatory vesicle and NF-κB pathway . Recent evidence has brought that nanoscale topographic modification of titanium substrates played a critical role in osseointegration . Data from Chauhan’s study had also suggested that there was a faster osseointegration with the strong interface on the surface of simvastatin-loaded titanium nanotubes implant surface . The above data thus provide a hint concerning the ‘nano scale-based’ work pattern of biomaterials in alleviating OIRR. Here, in our study, we successfully built a delivery complex, tFNAs-rGO, consisting of a 2D reduced graphene oxide (rGO) nanosheet loaded with a 3D nanoscale tetrahedral framework nucleic acid (tFNAs) (Fig. ). The synthesis of tFNAs-rGO using self-assembly technology is simpler and highly reproducible with a yield of approximately 90% compared with other materials . Most notably, the complex is relatively stable because the DNA tetrahedral structure is not easily degraded. Moreover, compared to other delivery platforms, rGO nanosheets not only have more outstanding carrying capacity of tFNAs due to their active surface properties, but their superior mechanical properties also make the delivery complex less susceptible to be damaged or removed under the intervention of mechanical force during tooth movement . In the Table S3, we discussed and summarized the characteristics of the tFNAs-rGO complex with previously published biomaterials. First, we validated the successful synthesis of tetrahedral tFNAs with negative charges through experiments such as AGE, AFM, and TEM. And tFNAs was uniformly loaded onto rGO nanosheets. Then, it confirmed that the tFNAs-rGO delivery complex had no significant biological toxicity by cell viability experiments in vitro. Moreover, the results from HE staining implied that tFNAs-rGO delivery complex would not influence the cellular morphology and tissue structure of organs. It was shown that the tFNAs-rGO delivery complex significantly reduced the region of root resorption and downregulated osteoclast activity in the peri-root tissue. Interestingly, we observed that the complex did not decrease the efficiency of tooth movement under stereomicroscope and 3D reconstruction of Micro-CT. The above results suggested that the tFNAs-rGO delivery complex could alleviate OIRR without affecting tooth movement. This means that, after the transformation and application of the tFNAs-rGO delivery complex, it is highly likely to solve the problems of orthodontists, such as having to suspend orthodontic treatment when root resorption occurs . Certainly, further work is needed to verify the feasibility and fully reveal the mechanism by which the tFNAs-rGO delivery complex may ameliorate OIRR. Previous studies demonstrated that root resorption like orthodontically induced root resorption (OIRR) was a complex and orchestrate pathological activity underlying a non-physiologic cellular activation and close crosstalk of numerous cells . So far, numerous in vitro and in vivo studies have delved into the molecular mechanisms of OIRR. Abnormalities of clastic cell adhesion, fusion and activation exacerbated the root resorption by the specific pathways such as RANKL/RANK/OPG signaling . Cementum had a reparative ability to mineralized tissue and was commonly regarded as an anti-resorptive barrier because of its mineral-remodeling process deficiency . Salman found that, as a “defense factor” for oxidative stress in cementum maintenance, superoxide dismutase 3 (SOD3) as a “defense” from oxidative stress strongly expressed in cementocytes and cementoblasts by proteomics . It was noteworthy that SOD3 was immunolocalized around apical third cementocytes and in cervical root cementoblasts, which were sensitive to root resorption. Based on the recent reports, the molecular pathway leading to root resorption is not likely to be one. Certainly, it is not dependent on a single cell population. There are several effector cells in the root resorption process, specifically, PDLCs, odontoclasts, osteoclasts, and macrophages . The current study identified that the enhanced Wnt5a-Ror2 signaling in PDLCs promoted the release of RANKL and partly led to odontoclast activity upregulation and final root resorption . The Chen’s lab reported that orthodontic force-induced BMAL1 in PDLCs was closely involved in controlling osteoclastic activities in alveolar bone remodeling . Here we showed that the tFNAs-rGO delivery complex boosted the ability of osteogenesis in PDLCs under in vitro osteogenic induction circumstance. Furthermore, compared with rGO, the tFNAs-rGO delivery complex exhibited a stronger promoting effect on osteogenic differentiation of PDLCs, illustrating in its more outstanding promotion of the mineralized nodules formation and upregulation of the expression of osteogenic related molecules. Consistent with Elhakim’s results, icariin was demonstrated potent pro-osteogenic property that promoted functional healing and reduced resorption of denuded roots in molar replantation rat . Therefore, we speculate that the tFNAs-rGO delivery complex may abate OIRR partly by promoting osteogenic differentiation of PDLCs around the roots. The critical influence of macrophages in root resorption has been widely recognized . Macrophages are mainly divided into M1 type and M2 type. M1-type macrophage is pro-inflammatory type and releases various inflammatory mediators, and drives osteoclastogenesis, whereas M2-type macrophages are anti-inflammatory cell subpopulation and support the deposition and mineralization of bone . Notably, our data also revealed that the tFNAs-rGO delivery complex reversed OIRR-enhanced M1/M2 macrophage ratio in mice and suppressed M1 and promoted M2 polarization in vitro. Meanwhile, our study showed that tFNAs-rGO evidently promoted the migration of RAW264.7 cells in transwell migration assay. Fang et al. proposed that the inflammatory monocytes homed in a CXCL12/CXCR4 axis-dependent manner and undergone M1 polarization in orthodontic root resorption, which were blunted by intraperitoneal injection of CXCR4 antagonist AMD3100 . Based on the above results, we hypothesized that there was a possibility that the tFNAs-rGO delivery complex facilitated the migration of macrophages to the site of root resorption, which then polarized to M2-phenotype protected macrophages. Concurrently, tFNAs-rGO could also inhibit the polarization of macrophages in the root resorption area towards M1 anti-inflammatory type, ultimately alleviating OIRR. Above all, we outlined a scheme for PDLCs, and macrophages mediated dual-functional tFNAs-rGO delivery complex remission of orthodontically induced root resorption. There are several recognized limitations to our study. First, in this study, we have demonstrated that the tFNAs-rGO delivery complex exerted dual functions of the osteogenic differentiation of PDLCs and the M2 phenotype polarization. However, we have not yet fully revealed the specific mechanism by which the tFNAs-rGO delivery complex affected these two cells, or whether there is crosstalk between PDLCs and macrophages in the OIRR treatment. Single-cell profiling will help to uncover the coordinated cell-cell interaction and the underlying regulation pathways. This will be a vital direction of future study. Second, there is a great unmet medical need for a nanoscale biological reagent to combat OIRR. Although we have preliminary confirmed that the feasibility of applying the tFNAs-rGO delivery complex to treat OIRR, it is just the first step toward the translation of our study into clinical application. Further investigation is needed on the appropriate tFNAs-rGO delivery complex dosage and administration using large-animal models. 

In conclusion, we successfully synthesized a promising nanoscale delivery complex, dual-functional tFNAs-rGO, which was applied to alleviate orthodontically induced root resorption by regulating PDLCs and macrophages (Fig. ). The prepared tFNAs were well-distributed onto the surface of rGO nanosheets. The dual-functional tFNAs-rGO delivery complex promoted the differentiation of PDLCs towards osteogenic direction and switched the macrophage polarity to the classically anti-inflammatory M2 phenotype. This study is the first to introduce nanosheets loading 3D nucleic acids for in OIRR therapy. Our findings provide further insight into a new tFNAs upgrading strategy for maintaining the biological functions and suggest a potential therapeutic candidate in the treatment of OIRR. 

Below is the link to the electronic supplementary material. Supplementary Material 1 

