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. 2022 May 31;30(10):3226–3240. doi: 10.1016/j.ymthe.2022.05.020

circRNA422 enhanced osteogenic differentiation of bone marrow mesenchymal stem cells during early osseointegration through the SP7/LRP5 axis

Ke Yu 1,2, Zhiwei Jiang 1,2, Xiaoyan Miao 1, Zhou Yu 1, Xue Du 1, Kaichen Lai 1, Ying Wang 1, Guoli Yang 1,∗
PMCID: PMC9552913  PMID: 35642253

Abstract

Circular RNAs (circRNAs) play an important role in biological activities, especially in regulating osteogenic differentiation of stem cells. However, no studies have reported the role of circRNAs in early osseointegration. Here we identified a new circRNA, circRNA422, from rat bone marrow mesenchymal stem cells (BMSCs) cultured on sandblasted, large-grit, acid-etched titanium surfaces. The results showed that circRNA422 significantly enhanced osteogenic differentiation of BMSCs with increased expression levels of alkaline phosphatase, the SP7 transcription factor (SP7/osterix), and lipoprotein receptor-related protein 5 (LRP5). Silencing of circRNA422 had opposite effects. There were two SP7 binding sites on the LRP5 promoter, indicating a direct regulatory relationship between SP7 and LRP5. circRNA422 could regulate early osseointegration in in vivo experiments. These findings revealed an important function of circRNA422 during early osseointegration. Therefore, circRNA422 may be a potential therapeutic target for enhancing implant osseointegration.

Keywords: circular RNAs, titanium implant, bone marrow mesenchymal stem cells, SP7/LRP5 axis, osseointegration

Graphical abstract

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Yu et al. identified a new circRNA, circRNA422, from rat BMSCs cultured on SLA titanium surfaces. circRNA422 interfering inhibited osteogenic differentiation of BMSCs through the SP7/LRP5 axis. circRNA422 overexpression promoted early osseointegration in vivo, indicating its potential therapeutic application for enhancing implant osseointegration.

Introduction

Circular RNAs (circRNAs) have been verified to play an important role in biological activities,1,2 including cell proliferation, self-renewal, and osteogenic differentiation of stem cells.3,4 Compared with other linear RNAs, circRNAs are endowed with high stability and a unique conformation in cells and tissues because of their covalently closed structure.5 Currently, the most intensively studied gene transfection objects are linear RNAs (miR-21, antimR-138, or siMIR31HG), which are assembled on a titanium surface to enhance osteogenic differentiation of stem cells.6, 7, 8, 9 However, linear RNAs are less stable with shorter half-lives than circRNAs.10 One study revealed that biomaterials could modulate differential expression of circRNAs during the osteogenic process of bone marrow mesenchymal stem cells (BMSCs).11 One group identified and constructed circRNA profiles on a titanium surface treated with mechanical attrition.12 The findings indicated the potential function of circRNAs in osseointegration. Recent research showed that circ_0008542 in osteoblast exosomes promotes osteoclast-induced bone resorption, leading to destruction of osseointegration.13 However, no studies have evaluated the positive role of circRNAs in early osseointegration.

circRNAs have been reported to modulate gene expression in the nucleus, function as microRNA (miRNA) sponges and protein scaffolds,14,15 sequester proteins,16 and more.17, 18, 19 The most widely investigated biological function of circRNAs is to serve as sponges of miRNAs to regulate key transcription factors in osteogenesis.20,21 The circRNA CDR1 acts as a sponge for miR-7, triggering upregulation of growth differentiation factor 5 expression and subsequent Smad1/5/8 expression to promote periodontal ligament stem cell (PDLSC) osteogenic differentiation.22 circRNA-vgll3 has been shown to directly sequester miR-326-5p to promote integrin 5 translation during osteogenic differentiation of adipose-derived mesenchymal stem cells (ADSCs).23 Therefore, it is feasible to explore the mechanism of circRNAs from the perspective of regulating osteogenic transcription factors during early osseointegration.

As a key transcription factor, the SP7 transcription factor (SP7/osterix) has been reported to be indispensable for osteogenesis.24, 25, 26 Current studies have focused on exploring various methods to enhance expression of SP7 to improve osseointegration. A previous study reported that direct injection of SP7 into peri-implant sites promotes osteogenic differentiation of BMSCs around the implant.27 Many researchers have developed various titanium modifications to upregulate expression of SP7.28,29 The effect of circRNA on expression of SP7 in BMSCs during early osseointegration has not been fully elucidated. SP7 has been shown to promote transcription of Colla1 by binding SP1 sites.30 There are many SP1 binding sites in the low-density lipoprotein receptor-related protein 5 (LRP5) promoter.31 SP7-expressing cells have been found to exhibit increased expression levels of Wnt10b and Axin2,32 key factors of the Wnt signaling pathway. This evidence suggested that SP7 might affect LRP5 to activate the Wnt signaling pathway. LRP5 has been found to maintain self-renewal and differentiation of stem cells.33, 34, 35 LRP5-modified BMSC sheet-implant complexes have been shown to promote osseointegration.36 circRNA422, originating from the exon of the KLHL8 gene has been reported to show upregulated expression during osteogenic differentiation of rat dental follicle cells.37 However, the interaction among circRNA422, SP7, and LRP5 in osteogenic differentiation remains unclear. Therefore, we attempted to identify circRNA422 associated with SP7 and propose that SP7 regulates expression of LRP5 during the process of osseointegration.

The aims of this study were to characterize a new circRNA (exon-derived circRNA422) and to investigate its function in osteogenic integration in vitro and in vivo.

Results

Characterization of BMSCs

Primary rat BMSCs effectively attached to the culture plate and exhibited a fibroblast-like and spindle-shaped morphology (Figure S1A). Colony formation assays with crystal violet staining showed that a representative colony-forming unit-fibroblast (CFU-F) contained spindle-shaped mesenchymal stem cells (MSCs) (Figure S1B). Flow cytometry analysis of passage 2 BMSCs showed that BMSCs were positive for the stemness markers CD29 (98.2%) and CD90 (95.0%) but did not express the hematopoietic stem cell markers CD34 (1.20%) and CD45 (2.24%) (Figures S1C−S1F).

Characterization of circRNA422 in BMSCs

The morphology of smooth titanium (Con_Ti group) and sandblasted, large-grit, acid-etched (SLA) titanium (SLA_Ti group) is shown in Figure 1A. In the Con_Ti group, the titanium surface had a very smooth topography with visible fine polishing lines. After SLA treatment, the surface was rough, and small holes had formed (approximately 1–2 μm in diameter) with pits. Hierarchical clustering revealed that circRNA422 expression was upregulated in the SLA_Ti group compared with the Con_Ti group, as shown in Figure 1B.

Figure 1.

Figure 1

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Characterization and expression of circRNA422 in BMSCs and the subcellular location of circRNA422

(A) Scanning electron microscopy (SEM) images of smooth and SLA Ti surfaces. Scale bars, 2 μm. (B) Hierarchical clustering revealed significantly differentially expressed circRNAs in the Con_Ti and SLA_Ti groups during osteogenic differentiation of BMSCs. (C) circRNA422 generated from the exon of the KLHL8 gene locus. (D) The PCR products of circRNA422 showed a single band in agarose gel electrophoresis. (E) Sanger sequencing results showed the backsplicing junction of circRNA422 in the PCR products. (F) The melting peak of circRNA422 divergent primers. (G) The results of RNase R digestion (n = 3, ∗p < 0.05). (H) Quantitative real-time PCR analysis of circRNA422 expression in BMSCs on the SLA Ti surface compared with that on the smooth Ti surface on osteogenic day 7 (n = 3, ∗p < 0.05). (I and J) PCR and quantitative real-time PCR analyses of circRNA422 expression in BMSCs on osteogenic days 3, 7, and 14 (n = 3, ∗p < 0.05). (K and L) Nucleoplasmic separation and FISH experiments showed that circRNA422 was more likely to be located in the cytoplasm of BMSCs. Scale bars, 20 μm. ∗p < 0.05.

circRNA422 expression has been reported to be upregulated during osteogenic differentiation of rat dental follicle cells.37 circRNA422 originates from the exon of the KLHL8 gene (Figure 1C). Divergent primers were designed according to the back-splicing junction of circRNA422. The polymerase chain reaction (PCR) products of circRNA422 showed a single band in agarose gel electrophoresis (Figure 1D) and contained the backsplicing junction of circRNA422 (Figure 1E). In Figure 1F, the melting curve of circRNA422 divergent primers showed a single peak. Figure 1G shows that circRNA422 resisted RNase R digestion. The expression levels of circRNA422 were upregulated in the SLA_Ti group compared with the Con_Ti group (Figure 1H). The PCR and quantitative real-time PCR results showed that the expression levels of circRNA422 gradually increased during osteogenic differentiation of BMSCs (Figures 1I and 1J). As demonstrated by the nucleoplasmic separation experiments and fluorescence in situ hybridization (FISH), circRNA422 was more likely to be located in the cytoplasm of BMSCs, as shown in Figures 1K and 1L.

circRNA422 facilitated BMSC proliferation and osteogenic differentiation by regulating expression of SP7 and LRP5

A circRNA422-overexpressing lentivirus (circRNA422+) and the control (Con510) as well as a circRNA422-interfering lentivirus (circRNA422-) and the control (Con313) were transfected into BMSCs to explore the function of circRNA422 during osteogenic differentiation of BMSCs. The experiments performed to determine a suitable multiplicity of infection (MOI) of the lentivirus for BMSCs are shown in Figure S2, and the results of flow cytometry indicated that transfection efficiency reached more than 80% in the MOI 30 group (Figure 2A). Therefore, an MOI of 30 was chosen for subsequent experiments.

Figure 2.

Figure 2

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Lentivirus transfection of circRNA422 in BMSCs

(A) In the MOI 30 group, the ratio of FITC-positive cells to live cells was 88.6%. (B and C) Quantitative real-time PCR and PCR analyses showed that circRNA422− and circRNA422+ significantly knocked down and upregulated expression of circRNA422 (n = 3, ∗p < 0.05). (D) Alamar blue analysis detecting the optical density (OD) value of the four groups (Con313, circRNA422−, Con510, and circRNA422+) showed that circRNA422− inhibited BMSC proliferation on day 7 and that circRNA422+ promoted BMSC proliferation on days 3 and 7 (n = 3, ∗p < 0.05).

As shown in Figure 2B, BMSCs showed approximately 26-fold upregulation of circRNA422 expression in the circRNA422+ group compared with the Con510 group. circRNA422 expression of BMSCs in the circRNA422− group was approximately 0.4-fold upregulated compared with the Con313 group. The PCR analysis shown in Figure 2C indicated similar effects of overexpressing and interfering lentiviruses on BMSCs.

On osteogenic days 3 and 7, alkaline phosphatase (ALP) staining was performed, and the color in the circRNA422− group was lighter than that in the Con313 group, as shown in Figure 3A. On osteogenic days 14 and 21, the osteocalcin (OCN) protein significantly decreased in the circRNA422− group (Figure 3B). Figure 3C shows that the mRNA expression levels of osteogenic genes, including ALP, OCN, LRP5, SP7, bone sialoprotein (BSP), and osteopontin (OPN) were suppressed by circRNA422− compared with Con313 on osteogenic days 3 and 7. The protein expression levels of ALP, LRP5, and SP7 were consistent with the quantitative real-time PCR results in Figure 3D.

Figure 3.

Figure 3

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circRNA422 interfering inhibited osteogenic differentiation of BMSCs

(A) ALP staining to evaluate the effect of circRNA422− on the ALP activity of BMSCs on osteogenic days 3 and 7. (B) Protein levels of OCN to evaluate the effect of circRNA422− on mineralization of BMSCs on osteogenic days 14 and 21. (C) Quantitative real-time PCR analysis showed that the osteogenic mRNA expressions levels of SP7, LRP5, ALP, OCN, BSP, and OPN were downregulated by circRNA422− compared with Con313 on osteogenic days 3 and 7 (n = 3, ∗p < 0.05). (D) WB analysis showed that the osteogenic protein expressions levels of ALP, LRP5, and SP7 were reduced by circRNA422− compared with Con313 on osteogenic days 3 and 7 (n = 3, ∗p < 0.05).

Figure 4A shows that the ALP staining in the circRNA422+ group was darker than that in the Con510 group. On osteogenic day 14, the OCN protein level showed no significant difference in the circRNA422+ group. On osteogenic day 21, the OCN protein significantly increased in the CircRNA422+ group, as shown in Figure 4B. As shown in Figure 4C, the mRNA expression levels of SP7 and OCN were not significantly increased in the circRNA422+ group on osteogenic day 3, but the mRNA expression levels of SP7, LRP5, ALP, OCN, BSP, and OPN were enhanced by circRNA422+ on osteogenic day 7. In Figure 4D, the protein expression levels of ALP, LRP5, and SP7 showed a similar trend to the quantitative real-time PCR results. These data demonstrate that circRNA422 regulated osteogenic differentiation of BMSCs by influencing expression of SP7 and LRP5.

Figure 4.

Figure 4

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circRNA422 overexpression promoted osteogenic differentiation of BMSCs

(A) ALP staining to evaluate the effect of circRNA422+ on the ALP activity of BMSCs on osteogenic days 3 and 7. (B) Protein levels of OCN to evaluate the effect of circRNA422+ on mineralization of BMSCs on osteogenic days 14 and 21. (C) Quantitative real-time PCR analysis showed that the osteogenic mRNA expressions levels of SP7, LRP5, ALP, OCN, BSP, and OPN were upregulated by circRNA422+ compared with Con510 on osteogenic day 7. The mRNA expression of SP7 and OCN was upregulated by circRNA422+ compared with Con510 on osteogenic day 3 (n = 3, ∗p < 0.05). (D) WB analysis showed the osteogenic protein expressions levels of ALP, LRP5, and SP7 on osteogenic days 3 and 7 (n = 3, ∗p < 0.05).

Identification of the SP7/LRP5 axis

To determine the possible relationship between SP7 and LRP5, lentiviral and adenoviral vectors were constructed to transfect BMSCs to alter the expression levels of SP7 and LRP5. The experiments to determine a suitable MOI for adenoviruses are shown in Figure S3, and the flow cytometry results indicated that the transfection efficiency reached more than 80% in the MOI 10 group. The mRNA expression levels of SP7 were downregulated by an LRP5-interfering lentivirus (LRP5−) and upregulated by an LRP5-overexpressing adenovirus (LRP5+) (Figure 5A). mRNA expression levels of LRP5 were downregulated by an SP7-interfering lentivirus (SP7−) and upregulated by an SP7-overexpressing adenovirus (SP7+) (Figure 5B). When BMSCs were cotransfected with LRP5+ and SP7− viruses, quantitative real-time PCR analysis revealed that the upregulated expression of ALP, Runt-related transcription factor 2 (Runx2), β-catenin, SP7, and LRP5 caused by LRP5+ was impaired by SP7− (Figure 5C). Similarly, the downregulated mRNA expression of these osteogenic genes caused by LRP5− was partially rescued by SP7+ (Figure 5D). Two potential binding sites of SP7 were predicted and confirmed in the LRP5 promoter by chromatin immunoprecipitation (ChIP) experiments (Figures 5E and 5F).

Figure 5.

Figure 5

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Identification of the SP7/LRP5 axis

(A) Quantitative real-time PCR analysis detecting the mRNA expression levels of SP7 and LRP5 on osteogenic day 7 showed that they were significantly promoted by LRP5+ and attenuated by LRP5− (n = 3, ∗p < 0.05). (B) Quantitative real-time PCR analysis detecting the mRNA expression of SP7 and LRP5 on osteogenic day 7 showed that they were significantly promoted by SP7+ and attenuated by SP7− (n = 3, ∗p < 0.05). (C) Quantitative real-time PCR analysis showing osteogenic mRNA expression of ALP, Runx2, β-catenin, SP7, and LRP5 on osteogenic day 7 after cotransfection of LRP5+ and SP7− in BMSCs (n = 3, ∗p < 0.05). (D) Quantitative real-time PCR analysis showing osteogenic mRNA expression of ALP, Runx2, β-catenin, SP7, and LRP5 on osteogenic day 7 after cotransfection of LRP5− and SP7+ in BMSCs (n = 3, ∗p < 0.05). (E) Prediction of binding sites using bioinformatics. (F) The results of the ChIP experiments.

Identification of successful ZsGreen1-labeled lentiviral vector transfection

In Vivo Imaging Ssystem (IVIS) Spectrum was used to verify the expression of ZsGreen1 (a green fluorescence protein) around the implants 14 days after implantation. However, there was a bright signal from the forelimb area because of autofluorescence from the fur that interfered with collection of signals. The ZsGreen1signals were difficult to detect even when the fur around the tibiae was removed in vivo, as shown in Figure 6A. Therefore, for detection of the signals, the rat tibiae were separated and scanned by the IVIS Spectrum. The results demonstrated that ZsGreen1 signals were detected in vitro using this method (Figure 6B).

Figure 6.

Figure 6

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Identification of lentiviruses at the peri-implant site in tibiae and fluorochrome labeling analysis of the biological function of circRNA422

(A) CCD images from the observation of ZsGreen1 signals in tibiae (right) in vivo after implantation at 14 days. No obvious ZsGreen1 signals were observed in tibiae with lentivirus injection and no lentivirus injection. (B) CCD images from the observation of ZsGreen1 signals in tibiae (right) in vitro after implantation at 14 days. The color indicates the bioluminescence intensity in photos per second per cm2 per steradian. (C) Images of fluorochrome labeling around the implant. The white arrow indicates the thickness of newly formed bone. Scale bars, 100 μm. (D) The MAR of the circRNA422− group was lower than that of the Con313 group, and the MAR of the circRNA422+ group was higher than that of the Con510 group (n = 6, ∗p < 0.05).

circRNA422 regulated early osseointegration in vivo

Calcein and alizarin were administered for fluorochrome labeling to indicate new bone deposition 1 or 5 weeks and 3 or 7 weeks after the implantation surgery, respectively. The distance between calcein- and alizarin-positive sites represented the thickness of newly formed bone around the implant. The mineral apposition rate (MAR) was used to measure the speed of new bone formation in the four groups. At 4 weeks, the MAR of the circRNA422+ group was higher than that of the Con510 group, and the MAR of the circRNA422− group was lower than that of the Con313 group (Figures 6C and 6D). However, there was no significant difference in MAR among these groups at 8 weeks (Figure S4).

Microcomputed tomography (micro-CT) analysis was performed to evaluate new bone formation around the implant at 4 and 8 weeks. The results at 4 weeks showed that the bone volume/total volume (BV/TV) and average trabecular thickness (Tb.Th) values in the circRNA422+ group were higher than in the Con510 group (p < 0.05), whereas those in the circRNA422− group were significantly lower than in the Con313 group (p < 0.05) (Figures 7A–7C). No significant difference was found in the number of trabeculae per millimeter (Tb.N) (Figure 7D). No significant difference was found in the micro-CT analysis at 8 weeks (Figures S5A–S5D).

Figure 7.

Figure 7

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Micro-CT and hard tissue section analysis of the biological function of circRNA422 in vivo

Samples and tissue sections from the 4 groups were observed and imaged after 4 weeks. (A) Three-dimensional reconstruction from four groups was observed and imaged after 4 weeks of implantation. Scale bars, 1 mm. (B and C) The BV/TV and Tb.Th values of the circRNA422− group were lower than those of the Con313 group, and the BV/TV and Tb.Th values of the circRNA422+ group were higher than those of the Con510 group (n = 6, ∗p < 0.05). (D) No significantly difference was found in Tb.N. (E) Images of methylene blue-acidic magenta dyeing. Methylene blue stains collagen fibers, and acidic magenta stains new bone around the implant. Scale bars, 200 μm. (F and G) The BIC and BV/TV values of the circRNA422− group were lower than those of the Con313 group, and the BIC and BV/TV values of the circRNA422+ group were higher than those of the Con510 group (n = 6, ∗p < 0.05).

Methylene blue-acidic magenta dyeing of hard tissue sections is another important tool to evaluate osseointegration. Methylene blue stains collagen fibers, and acidic magenta stains new bone around the implant.38 At 4 weeks, the circRNA422+ group exhibited a higher bone-implant-contacting area percentage (BIC) and bone BV/TV, and the circRNA422− group had a lower BIC and BV/TV (Figures 7F and 7G). At 8 weeks, the BIC value in the circRNA422− group was significantly lower than in the Con313 group (p < 0.05). No significant difference was found in other values at 8 weeks (Figures S5F and S5G). These data suggested that circRNA422 regulated early osseointegration in vivo.

The mechanism for the function of circRNA422 in implant osseointegration is summarized in Figure 8. circRNA422, identified from SLA titanium surfaces, could regulate osteogenic differentiation of BMSCs by upregulating the expression levels of SP7 and LRP5, facilitating early new bone formation around the implant.

Figure 8.

Figure 8

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Mechanism of circRNA422 regulating BMSC osteogenic differentiation in early osseointegration

Discussion

Emerging evidence has shown that circRNAs play an important role in osteogenic differentiation of stem cells and bone healing.39,40 However, no studies have evaluated the role of circRNAs in early osseointegration or their underlying mechanisms. Our study identified a new circRNA, circRNA422, and characterized its loop structure. We demonstrated that there were two binding sites of SP7 on the LRP5 promoter. We presented evidence showing that circRNA422 could promote osteogenic differentiation of BMSCs through the SP7/LRP5 axis. Local administration of circRNA422 markedly enhanced early osseointegration.

In the present study, we locally injected a circRNA422 lentivirus into the implant holes, as in a previous article.41 We successfully promoted new bone formation around the implant, demonstrating that circRNAs could be potential therapeutic strategies for bone regeneration. A previous study also verified that circRNA hsa_circ_0074834-modified BMSCs could improve BMSC osteogenesis when implanted into a femoral monocortical defect model.42 This circRNA-based therapeutic agent has been validated in other stem cells. ADSCs transfected with the circFOXP1 overexpression vector were encapsulated in collagen-based hydrogels, and then the complexes were used to promote heterotopic bone formation.43 However, based on circRNAs, multiple therapeutic agents could promote osseointegration, which requires further investigation.

Animal experiments in this study showed that circRNA422 could promote implant osseointegration at 4 weeks but had no significant effect at 8 weeks. The reason might be that the bone formation rate in rats could be achieved in 4 weeks, much faster than the implants in humans.44 A prior published study reported similar results that the peri-implant BV (thickness) between the zoledronic acid group and the control groups existed significant difference during the early stage of osseointegration but showed no significant difference during the late stage of osseointegration.45 Based on evidence from in vivo and in vitro experiments, we concluded that circRNA422 enhanced early osseointegration.

Other known osteogenic factors, such as BMP, were used with the style of mRNA or protein.46 The mRNA and protein are still limited in clinical applications because of the short half-life or robust release of BMP2.47 However, circRNA has been reported to have the advantages of high sequence conservation, abundant quantity, and higher stability.48,49 Therefore, circRNA have become a potential therapeutic target in future applications.

circRNAs modulate gene expression in the nucleus, act as sponges for miRNAs, serve as scaffolds for circRNA-protein complexes, and even act as templates for translation.2 circRNAs have been shown to play a vital role in differentiation of stem cells primarily by regulating expression of key transcription factors. SP7 is a zinc-finger transcription factor that functions in osteogenesis and enhances the proliferation and osteogenic potential of BMSCs.24 SP7 expression was upregulated on the different titanium surfaces and was related to titanium surface-mediated osteogenesis.50 Thus, our study evaluated mRNA and protein expression of SP7 during circRNA422-mediated osteogenic differentiation of BMSCs and demonstrated that circRNA422 could upregulate the expression levels of SP7. Several studies indicated that circRNA_0062582 and circ_0000020 promote levels of SP7 during osteogenic differentiation of BMSCs.21,51 Other transcription factors, such as Runx2, might be also regulated by some circRNAs.52,53

Recent studies have revealed that mRNA levels of SP7 were reduced in mice with heterozygous deletion of the LRP5 gene,22 and expression of SP7 was higher in LRP5 ACT mice54 (a transgenic mouse with a high bone mass phenotype because of expression of the human G1717V mutation) than in wild-type mice.55 Our study indicated that LRP5 positively influenced expression of SP7. Regarding regulation of LRP5 by SP7, it has been documented that SP7-expressing cells can induce the Wnt signaling response to regulate their proliferation and differentiation.32 This study has extended the function of SP7 to regulate expression of LRP5, which is the key to Wnt signaling. As expected, SP7 overexpression markedly increased mRNA expression of LRP5 and SP7 interfering significantly decreased mRNA expression of LRP5 (Figure 5B). We predicted and verified two binding sites of SP7 on the LRP5 promoter, indicating that SP7 might regulate transcription of LRP5 in BMSCs. In this study, the expression levels of SP7 and LRP5 were regulated by the expression levels of circRNA422 in BMSCs. These results demonstrated that bidirectional regulation between SP7 and LRP5 was involved in circRNA422-mediated osteogenic differentiation of BMSCs.

Successful transfection of lentiviruses into peri-implant sites was the focus of our animal experiments. In this study, the ZsGreen1 (a green fluorescent protein) gene was used for identification of lentiviral transfection, and we found that the ZsGreen1 signals could not be detected directly in vivo using the IVIS Spectrum. Compared with the mouse model,41 the muscles, skin, and fur of rats were thicker, which might obscure the ZsGreen1 signals. Thus, in vitro detection was utilized to track lentiviral transfection. Luciferase signals have been detected in rat implant models in vivo using the IVIS Spectrum.56 In future studies, a luciferase lentivirus will be used to track successful target-circRNA transfection in rat tibiae.

In this study, a new circRNA, circRNA422, was identified from BMSCs cultured on SLA titanium surfaces and successfully characterized using a series of experiments. circRNA422 enhanced the osteogenic process by promoting differentiation of BMSCs in vitro and in vivo. However, the underlying mechanisms regarding material surface morphology and the properties influencing circRNA profiles need to be explored in further studies.

Conclusion

circRNA422 had a positive effect on osteogenic differentiation of BMSCs through the SP7/LRP5 axis. Our research indicated that circRNA422-overexpressing BMSCs showed stronger proliferation and osteogenic differentiation abilities than the controls. Local administration of circRNA422 enhanced early osseointegration. circRNA422 might be a potential target for enhancing implant osseointegration.

Materials and methods

BMSC isolation and culture

This study was approved by the Institutional Animal Care and Use Committee of Zhejiang University, Hangzhou, China. Four-week-old Sprague-Dawley (SD) rats were provided by the Shanghai Animal Experimental Center. Isolation and culture of BMSCs were performed as described previously.56 BMSCs at passage 2 were used for subsequent experiments. BMSCs were cultured in growth medium containing alpha-modified minimum essential media (α-MEM ; Gibco, USA), 10% fetal bovine serum (Gibco, Australia), 0.272 g/L L-glutamine (Gibco, USA), and 1% penicillin-streptomycin (Gibco, USA).

Colony formation assay

For evaluation of colony formation efficiency, passage 1 BMSCs were cultured at a density of 2 × 103 cells in a 6-cm dish. After culture for 7 days, the BMSCs were fixed in 4% paraformaldehyde (PFA; Beyotime, Shanghai, China) and then stained with crystal violet (Beyotime) for 30 min. Aggregates of 50 or more BMSCs were considered CFU-F colonies and recorded using a phase-contrast microscope (CKX41, Olympus, Japan).

Flow cytometry

Passage 2 BMSCs at a density of 2 × 106 cells/mL were suspended in ice-cold phosphate-buffered saline (PBS) and subsequently incubated with the following antibodies: anti-CD34 (ab81289; Abcam, Cambridge, MA), anti-CD45 (ab10558, Abcam), fluorescein isothiocyanate (FITC)-conjugated immunoglobulin G (IgG) H&L (ab6717, Abcam), anti-CD29 (102208; BioLegend, San Diego, CA), and anti-CD90 (17-0900-82; eBioscience, San Diego, CA). After the cells were washed, they were identified using a flow cytometer (FACScalibur, BD Biosciences, USA), and the data were analyzed using FlowJo 10 software (TreeStar, Ashland, OR). Each sample was evaluated in triplicate.

Sample collection

Smooth and SLA titanium (850-mm diameter and 2-mm-thick sheets of grade 2 unalloyed titanium [Ti]) were provided by Guangci (Ningbo, China). For identification of qualified Ti, the structure of the Ti surface was observed under a field-scanning electron microscope (FSEM; SU8010, Hitachi, Japan).

BMSCs (3 × 106) were plated on the smooth and SLA Ti surfaces in 100-mm culture dishes. BMSCs were induced by osteogenic differentiation medium (ODM), which included growth medium, 10 nM β-glycerophosphate (Sigma-Aldrich, St. Louis, MO), 50 μg/mL L-ascorbic acid (Sigma-Aldrich), and 100 nM dexamethasone (Sigma-Aldrich), for 7 days. The ODM was changed every 2 days.

circRNA microarray assays

Total RNA used for circRNA microarray assays was extracted from BMSCs using TRIzol reagent (Invitrogen, Carlsbad, CA). The sequencing library of circRNAs was constructed by LC Bio (China). Differentially expressed circRNAs were identified by | log2 (fold change) | > 1 and p < 0.05.

Lentivirus and adenovirus construction

The rat circRNA422 gene is located on chromosome 14. The oligonucleotide sequence was synthesized as indicated in Table S1. Lentiviruses of Con313, circRNA422−, Con510, and circRNA422+ were constructed by GeneChem (Shanghai, China). BMSCs were transfected with the corresponding lentiviruses (Con313, circRNA422−, and Con510, circRNA422+), and 40 μL/mL transfection reagent P (GeneChem, Shanghai, China) was added.

For further analysis of the regulatory relationship between SP7 and LRP5, interfering lentiviruses (lentivirus negative control group; LVNC, SP7−, and LRP5−) were constructed by GenePharma (Shanghai, China), and overexpressing adenoviruses (adenovirus negative control group; ADNC, SP7+, and LRP5+) were designed by Vigenebio (Shandong, China). The cloning capacity of lentiviral vectors has been shown to be limited, but adenoviral vectors can carry more gene fragments.57 It is difficult for lentiviral vectors to carry large genes, and LRP5 is a representative large gene of 4,803 bp. Thus, adenoviruses were used to upregulate LRP5 expression. Similar to the procedure for upregulation of LRP5 expression, adenoviruses were also used for upregulation of SP7 expression. The transfection efficiency of lentiviruses or adenoviruses was demonstrated by detection of ZsGreen1 or GFP by fluorescence microscopy and flow cytometry, respectively. The interfering fragment sequences used are shown in Table S1.

Agarose gel electrophoresis

Five hundred nanograms of RNA was used to synthesize cDNA with the PrimeScript RT reagent kit (TaKaRa, Kyoto, Japan). After amplification, cDNA was subjected to 1.5%–2% agarose gels to detect its PCR products and analyzed by Sanger sequencing.

FISH

The specific FISH probe for circRNA422 was purchased from RiboBio (Guangzhou, China). BMSCs were cultured in 24-well plates at a density of 2 × 104 cells/well overnight. BMSCs were fixed in 4% PFA for 15 min. After full hydration, a 30% H2O2 and pure methanol mixture (1:9) was added for 10 min. Then 0.2 mol/L hydrochloric acid was dropped onto the slide for 15 min at room temperature. Proteinase K was added and incubated in a molecular hybridization meter at 37°C for 20 min. Then the cells were incubated with 500 ng/mL CY3-labeled probe (circRNA422, U6, and 18S) in hybridization buffer at 37°C overnight. The 18S gene is a member of the rRNA gene family and one of the most commonly used FISH markers.58 Thus, 18S is always used as the housekeeping gene for FISH experiments. The nuclei were stained with 4,6-diamidino-2-phenylindole (DAPI; Beyotime), and BMSCs were imaged under confocal microscopy (Carl Zeiss, Germany).

Nucleoplasmic separation experiments

BMSCs (1 × 106) were collected and washed in PBS. Then the cells were treated with cell fractionation buffer and cell disruption buffer in sequence according to the PARIS kit (Invitrogen). Finally, the subcellular fractions of the cytoplasm and nucleus were individually separated. The expression levels of circRNA422, U6, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were analyzed by quantitative real-time PCR.

ALP staining and OCN quantitative detection analysis

BMSCs were cultured in 12-well plates transfected with the corresponding lentiviral vectors. On osteogenic days 3 and 7, BMSCs were fixed in 4% PFA and stained with an ALP staining kit (Beyotime). A rat OCN ELISA kit (Elabscience, Wuhan, China) was used to detect release of OCN on osteogenic days 14 and 21.

Quantitative real-time PCR

Quantitative real-time PCR was performed to detect the expression levels of circRNA and mRNA using TB Green PCR Mix (TaKaRa) in the 384 Real-Time PCR System (Bio-Rad, USA). After normalization to GAPDH for mRNA and circRNA, the expression levels of RNA were presented in the form of relative fold change to the control. The relative fold changes in circRNA422 expression of BMSCs on SLA Ti were compared with expression of circRNA422 on smooth Ti on day 7. The relative fold changes in circRNA422 expression were detected on days 7 and 14 and compared with expression of circRNA422 on day 3. BMSCs were cultured in 6-well plates transfected with the corresponding lentiviral vectors, and osteogenic genes were detected on osteogenic days 3 and 7. mRNA expression of the osteogenic genes ALP, Runx2, β-catenin, SP7, and LRP5 was detected on osteogenic day 7 in the six groups: LVNC ADNC, LRP5− ADNC, LRP5− SP7+, ADNC LVNC, LRP5+ LVNC, and LRP5+ SP7−. For the nucleoplasmic separation experiments, GAPDH was used as an internal reference for cytoplasmic RNA, and U6 was used as an internal reference for nuclear RNA. All primers for related mRNAs and circRNA422 are listed in Table S2.

Protein extraction and western blot (WB)

Radio Immunoprecipitation Assay lysis buffer (RIPA; Beyotime) and 1 μL/mL protease inhibitor cocktail (Beyotime) were used for protein extraction on osteogenic days 3 and 7. Electrophoresis, membrane transfer, and blocking were performed as described previously.36 The primary antibodies used in the experiments included antibodies against ALP (DF6225; Affinity Biosciences, Jiangsu, China), LRP5 (D80F2; Cell Signaling Technology, Danvers, MA), SP7 (ab22552; Abcam), and GAPDH (60004-1; Proteintech, Rosemont, IL) (diluted 1:1,000). Next, the membranes were incubated with the appropriate secondary antibodies (Sigma-Aldrich). WBs were viewed using the Odyssey image scan system (Bio-Rad).

ChIP experiments

ChIP experiments were performed using the Agarose ChIP kit (Thermo Fisher Scientific, Waltham, MA) and Chromatin Prep Module (Thermo Fisher Scientific). DNA from BMSCs was obtained, purified, and subjected to quantitative real-time PCR.

Animal experiments

All institutional and national guidelines for the care and use of animals were followed. The in vivo study was approved by the Institutional Animal Care and Use Committee of Zhejiang University (grant 20220054, Hangzhou, China). A total of 24 SD rats were divided into four groups as follows: Con313, circRNA422−, Con510, and circRNA422+. The SLA implants used in this study were provided by Guangci and had a length of 5.0 mm and diameter of 2.2 mm. According to our previous study,36 the implant cavity was prepared on the tibiae approximately 7 mm below the knee joints of SD rats by drilling with a low rotational speed (500 rpm) under cooled sterile saline. Then four lentiviral vectors (1 × 109 transducing units/mL) were injected equally in a volume of 2 μL, and the SLA implants were press fitted into the prepared holes. After the operation, the rats were injected intramuscularly with penicillin daily for 3 days. The rats were given calcein (8 mg/kg, subcutaneous injection) 1 or 5 weeks after surgery and alizarin complexone (30 mg/kg, subcutaneous injection) 3 or 7 weeks after surgery. All animals were euthanized 4 weeks and 8 weeks after surgery to compare the osseointegration ability of the different groups.

Identification of lentiviral vector transfection

To determine whether the lentiviral vector could be successfully transfected into peri-implant sites, the IVIS Spectrum imaging system (PerkinElmer) was applied to detect expression of ZsGreen1 (absorbance of 465 nm) in the transfection group 14 days after local lentiviral vector injection in vivo. For in vitro observation, the tibiae were dissected from rats 14 days after surgery, and the samples were examined using IVIS and imaged with a charge-coupled device (CDD) camera.

Evaluation of circRNA422 function in vivo

Four and eight weeks after lentivirus injection and implant placement, tibiae with implants were obtained and fixed in 4% PFA for 24 h. There were 5 samples at 8 weeks because 2 rats died during the experiments. A 0.5-mm-radius circle from the implant surface was defined as the region of interest (ROI) and scanned with micro-CT (mCT-100; Scanco Medical, Switzerland). BV/TV, Tb.Th, and Tb.N were evaluated.

After micro-CT analysis, the samples were cut into slices along the long axis of the implants. The slices were observed under confocal microscopy (Carl Zeiss), and the MAR (μm/day) was recorded. Then the slices were stained with methylene blue-acidic magenta dye. Images of the implant and peri-implant bone tissues were acquired under a microscope (Olympus). The percentage of BV/TV on the interior of the threads and BIC in the threads along the surface of the implant were measured.

Statistical analysis

From 3 independent experiments, all data are expressed as the mean ± standard deviation. Statistical calculations were conducted using the SPSS 19.0 software package (IBM, Armonk, NY). Student’s t tests (for comparisons between two groups) and one-way analysis of variance with Bonferroni’s post hoc test (for multiple comparisons) were performed in this study. Differences were considered significant when p < 0.05.

Acknowledgments

This work was supported by the National Natural Science Foundation of China (grant 81901051); the Health Department of Zhejiang Province Fund, China (grant 2022KY872); and the Fundamental Research Funds for the Central Universities (2020FZZX008-03).

Author contributions

G.Y., K.Y., and Z.J. conceived the study. X.M. and Z.Y. developed the methodology. X.D. and K.L. acquired, analyzed, and interpreted data. Y.W., K.Y., and Z.J. performed statistical analysis and writing. All authors revised, read, and approved the final paper.

Declaration of interests

The authors declared no competing interests.

Footnotes

Supplemental information can be found online at https://doi.org/10.1016/j.ymthe.2022.05.020.

Supplemental information

Document S1. Figures S1–S5 and Tables S1 and S2
mmc1.pdf (4.5MB, pdf)
Document S2. Article plus supplemental information
mmc2.pdf (8.1MB, pdf)

References

  • 1.Goodall G.J., Wickramasinghe V.O. RNA in cancer. Nat. Rev. Cancer. 2021;21:22–36. doi: 10.1038/s41568-020-00306-0. [DOI] [PubMed] [Google Scholar]
  • 2.Chen L.L. The expanding regulatory mechanisms and cellular functions of circular RNAs. Nat. Rev. Mol. Cell Biol. 2020;21:475–490. doi: 10.1038/s41580-020-0243-y. [DOI] [PubMed] [Google Scholar]
  • 3.Chen L., Kong R., Wu C., Wang S., Liu Z., Liu S., Li S., Chen T., Mao C., Liu S. Circ-MALAT1 Functions as both an mrna translation brake and a microRNA sponge to promote self-renewal of hepatocellular cancer stem cells. Adv. Sci. (Weinh) 2020;7:1900949. doi: 10.1002/advs.201900949. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 4.Wang Y., Jiang Z., Yu M., Yang G. Roles of circular RNAs in regulating the self-renewal and differentiation of adult stem cells. Differentiation. 2020;113:10–18. doi: 10.1016/j.diff.2020.03.001. [DOI] [PubMed] [Google Scholar]
  • 5.Qu S., Yang X., Li X., Wang J., Gao Y., Shang R., Sun W., Dou K., Li H. Circular RNA: a new star of noncoding RNAs. Cancer Lett. 2015;365:141–148. doi: 10.1016/j.canlet.2015.06.003. [DOI] [PubMed] [Google Scholar]
  • 6.Geng Z., Yu Y., Li Z., Ma L., Zhu S., Liang Y., Cui Z., Wang J., Yang X., Liu C. miR-21 promotes osseointegration and mineralization through enhancing both osteogenic and osteoclastic expression. Mater. Sci. Eng. C Mater. Biol. Appl. 2020;111:110785. doi: 10.1016/j.msec.2020.110785. [DOI] [PubMed] [Google Scholar]
  • 7.Geng Z., Wang X., Zhao J., Li Z., Ma L., Zhu S., Liang Y., Cui Z., He H., Yang X. The synergistic effect of strontium-substituted hydroxyapatite and microRNA-21 on improving bone remodeling and osseointegration. Biomater. Sci. 2018;6:2694–2703. doi: 10.1039/c8bm00716k. [DOI] [PubMed] [Google Scholar]
  • 8.Wu K., Liu M., Li N., Zhang L., Meng F., Zhao L., Liu M., Zhang Y. Chitosan-miRNA functionalized microporous titanium oxide surfaces via a layer-by-layer approach with a sustained release profile for enhanced osteogenic activity. J. Nanobiotechnol. 2020;18:127. doi: 10.1186/s12951-020-00674-7. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 9.Huang Y., Zheng Y., Xu Y., Li X., Zheng Y., Jia L., Li W. Titanium surfaces functionalized with siMIR31HG promote osteogenic differentiation of bone marrow mesenchymal stem cells. ACS Biomater. Sci. Eng. 2018;4:2986–2993. doi: 10.1021/acsbiomaterials.8b00432. [DOI] [PubMed] [Google Scholar]
  • 10.Wesselhoeft R.A., Kowalski P.S., Anderson D.G. Engineering circular RNA for potent and stable translation in eukaryotic cells. Nat. Commun. 2018;9:2629. doi: 10.1038/s41467-018-05096-6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 11.Zhong W., Li X., Pathak J.L., Chen L., Cao W., Zhu M., Luo Q., Wu A., Chen Y., Yi L., et al. Dicalcium silicate microparticles modulate the differential expression of circRNAs and mRNAs in BMSCs and promote osteogenesis via circ_1983-miR-6931-Gas7 interaction. Biomater. Sci. 2020;8:3664–3677. doi: 10.1039/d0bm00459f. [DOI] [PubMed] [Google Scholar]
  • 12.Zhu S., Zhu Y., Wang Z., Liang C., Cao N., Yan M., Gao F., Liu J., Wang W. Bioinformatics analysis and identification of circular RNAs promoting the osteogenic differentiation of human bone marrow mesenchymal stem cells on titanium treated by surface mechanical attrition. PeerJ. 2020;8:e9292. doi: 10.7717/peerj.9292. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13.Wang W., Qiao S.C., Wu X.B., Sun B., Yang J.G., Li X., Zhang X., Qian S.J., Gu Y.X., Lai H.C. Circ_0008542 in osteoblast exosomes promotes osteoclast-induced bone resorption through m6A methylation. Cell Death Dis. 2021;12:628. doi: 10.1038/s41419-021-03915-1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 14.Hansen T.B., Jensen T.I., Clausen B.H., Bramsen J.B., Finsen B., Damgaard C.K., Kjems J. Natural RNA circles function as efficient microRNA sponges. Nature. 2013;495:384–388. doi: 10.1038/nature11993. [DOI] [PubMed] [Google Scholar]
  • 15.Du W.W., Yang W., Liu E., Yang Z., Dhaliwal P., Yang B.B. Foxo3 circular RNA retards cell cycle progression via forming ternary complexes with p21 and CDK2. Nucleic Acids Res. 2016;44:2846–2858. doi: 10.1093/nar/gkw027. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16.Abdelmohsen K., Panda A.C., Munk R., Grammatikakis I., Dudekula D.B., De S., Kim J., Noh J.H., Kim K.M., Martindale J.L., Gorospe M. Identification of HuR target circular RNAs uncovers suppression of PABPN1 translation by CircPABPN1. RNA Biol. 2017;14:361–369. doi: 10.1080/15476286.2017.1279788. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17.Zang J., Lu D., Xu A. The interaction of circRNAs and RNA binding proteins: an important part of circRNA maintenance and function. J. Neurosci. Res. 2020;98:87–97. doi: 10.1002/jnr.24356. [DOI] [PubMed] [Google Scholar]
  • 18.Huang A., Zheng H., Wu Z., Chen M., Huang Y. Circular RNA-protein interactions: functions, mechanisms, and identification. Theranostics. 2020;10:3503–3517. doi: 10.7150/thno.42174. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 19.Yang Y., Fan X., Mao M., Song X., Wu P., Zhang Y., Jin Y., Yang Y., Chen L.L., Wang Y., et al. Extensive translation of circular RNAs driven by N(6)-methyladenosine. Cell Res. 2017;27:626–641. doi: 10.1038/cr.2017.31. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 20.Ji F., Pan J., Shen Z., Yang Z., Wang J., Bai X., Tao J. The Circular RNA circRNA124534 promotes osteogenic differentiation of human dental pulp stem cells through modulation of the miR-496/β-catenin pathway. Front. Cell Dev. Biol. 2020;8:230. doi: 10.3389/fcell.2020.00230. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 21.Li F., Wu H., Zou G., Cang D., Shen Y. Circular RNA_0062582 promotes osteogenic differentiation of human bone marrow mesenchymal stem cells via regulation of microRNA-145/CBFB axis. Bioengineered. 2021;12:1952–1963. doi: 10.1080/21655979.2021.1921553. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22.Li X., Zheng Y., Zheng Y., Huang Y., Zhang Y., Jia L., Li W. Circular RNA CDR1as regulates osteoblastic differentiation of periodontal ligament stem cells via the miR-7/GDF5/SMAD and p38 MAPK signaling pathway. Stem Cell Res. Ther. 2018;9:232. doi: 10.1186/s13287-018-0976-0. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 23.Zhang D., Ni N., Wang Y., Tang Z., Gao H., Ju Y., Sun N., He X., Gu P., Fan X. CircRNA-vgll3 promotes osteogenic differentiation of adipose-derived mesenchymal stem cells via modulating miRNA-dependent integrin α5 expression. Cell Death Differ. 2021;28:283–302. doi: 10.1038/s41418-020-0600-6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 24.Tu Q., Valverde P., Chen J. Osterix enhances proliferation and osteogenic potential of bone marrow stromal cells. Biochem. Biophys. Res. Commun. 2006;341:1257–1265. doi: 10.1016/j.bbrc.2006.01.092. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25.Wu L., Wu Y., Lin Y., Jing W., Nie X., Qiao J., Liu L., Tang W., Tian W. Osteogenic differentiation of adipose derived stem cells promoted by overexpression of osterix. Mol. Cell Biochem. 2007;301:83–92. doi: 10.1007/s11010-006-9399-9. [DOI] [PubMed] [Google Scholar]
  • 26.Chia W., Liu J., Huang Y.G., Zhang C. A circular RNA derived from DAB1 promotes cell proliferation and osteogenic differentiation of BMSCs via RBPJ/DAB1 axis. Cell Death Dis. 2020;11:372. doi: 10.1038/s41419-020-2572-3. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 27.Xu B., Zhang J., Brewer E., Tu Q., Yu L., Tang J., Krebsbach P., Wieland M., Chen J. Osterix enhances BMSC-associated osseointegration of implants. J. Dent Res. 2009;88:1003–1007. doi: 10.1177/0022034509346928. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 28.Siqueira R., Ferreira J.A., Rizzante F.A.P., Moura G.F., Mendonça D.B.S., de Magalhães D., Cimões R., Mendonça G. Hydrophilic titanium surface modulates early stages of osseointegration in osteoporosis. J. Periodontal Res. 2021;56:351–362. doi: 10.1111/jre.12827. [DOI] [PubMed] [Google Scholar]
  • 29.Jann J., Drevelle O., Lauzon M.A., Faucheux N. Adhesion, intracellular signalling and osteogenic differentiation of mesenchymal progenitor cells and preosteoblasts on poly(epsilon)caprolactone films functionalized by peptides derived from fibronectin and/or BMP-9. Mater. Sci. Eng. C Mater. Biol. Appl. 2020;114:111088. doi: 10.1016/j.msec.2020.111088. [DOI] [PubMed] [Google Scholar]
  • 30.Niu P., Zhong Z., Wang M., Huang G., Xu S., Hou Y., Yan Y., Wang H. Zinc finger transcription factor Sp7/Osterix acts on bone formation and regulates col10a1a expression in zebrafish. Sci. Bull. 2017;62:174–184. doi: 10.1016/j.scib.2017.01.009. [DOI] [PubMed] [Google Scholar]
  • 31.Li J., Yang Y., Jiang B., Zhang X., Zou Y., Gong Y. Sp1 and KLF15 regulate basal transcription of the human LRP5 gene. BMC Genet. 2010;11:12. doi: 10.1186/1471-2156-11-12. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 32.Tan S.H., Senarath-Yapa K., Chung M.T., Longaker M.T., Wu J.Y., Nusse R. Wnts produced by osterix-expressing osteolineage cells regulate their proliferation and differentiation. Proc. Natl. Acad. Sci. U S A. 2014;111:E5262–E5271. doi: 10.1073/pnas.1420463111. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 33.Yan K.S., Janda C.Y., Chang J., Zheng G.X.Y., Larkin K.A., Luca V.C., Chia L.A., Mah A.T., Han A., Terry J.M., et al. Non-equivalence of Wnt and R-spondin ligands during Lgr5(+) intestinal stem-cell self-renewal. Nature. 2017;545:238–242. doi: 10.1038/nature22313. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 34.Esen E., Chen J., Karner C.M., Okunade A.L., Patterson B.W., Long F. WNT-LRP5 signaling induces Warburg effect through mTORC2 activation during osteoblast differentiation. Cell Metab. 2013;17:745–755. doi: 10.1016/j.cmet.2013.03.017. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 35.Liu J., Cui Z., Wang F., Yao Y., Yu G., Liu J., Cao D., Niu S., You M., Sun Z., et al. Lrp5 and Lrp6 are required for maintaining self-renewal and differentiation of hematopoietic stem cells. FASEB J. 2019;33:5615–5625. doi: 10.1096/fj.201802072r. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 36.Jiang Z., Yu K., Feng Y., Zhang L., Yang G. An effective light activated TiO2 nanodot platform for gene delivery within cell sheets to enhance osseointegration. Chem. Eng. J. 2020;402:126170. doi: 10.1016/j.cej.2020.126170. [DOI] [Google Scholar]
  • 37.Du Y., Li J., Hou Y., Chen C., Long W., Jiang H. Alteration of circular RNA expression in rat dental follicle cells during osteogenic differentiation. J. Cell Biochem. 2019;120:13289–13301. doi: 10.1002/jcb.28603. [DOI] [PubMed] [Google Scholar]
  • 38.Zhang D., Liu W., Wu X., He X., Lin X., Huang W. Novel nano-hydroxyapatite/polyurethane composite scaffold in the treatment of chronic osteomyelitis. Zhongguo Xiu Fu Chong Jian Wai Ke Za Zhi. 2018;32:880–886. doi: 10.7507/1002-1892.201802049. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 39.Yuan Y., Jiang Y., Wang B., Guo Y., Gong P., Xiang L. Deficiency of calcitonin gene-related peptide affects macrophage polarization in osseointegration. Front. Physiol. 2020;11:733. doi: 10.3389/fphys.2020.00733. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 40.Peng W., Zhu S., Chen J., Wang J., Rong Q., Chen S. Hsa_circRNA_33287 promotes the osteogenic differentiation of maxillary sinus membrane stem cells via miR-214-3p/Runx3. Biomed. Pharmacother. 2019;109:1709–1717. doi: 10.1016/j.biopha.2018.10.159. [DOI] [PubMed] [Google Scholar]
  • 41.Xiang L., Ma L., Wei N., Wang T., Yao Q., Yang B., Xiong Y., Wu Y., Gong P. Effect of lentiviral vector overexpression α-calcitonin gene-related peptide on titanium implant osseointegration in α-CGRP-deficient mice. Bone. 2017;94:135–140. doi: 10.1016/j.bone.2015.08.009. [DOI] [PubMed] [Google Scholar]
  • 42.Ouyang Z., Tan T., Zhang X., Wan J., Zhou Y., Jiang G., Yang D., Guo X., Liu T. CircRNA hsa_circ_0074834 promotes the osteogenesis-angiogenesis coupling process in bone mesenchymal stem cells (BMSCs) by acting as a ceRNA for miR-942-5p. Cell Death Dis. 2019;10:932. doi: 10.1038/s41419-019-2161-5. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 43.Shen W., Sun B., Zhou C., Ming W., Zhang S., Wu X. CircFOXP1/FOXP1 promotes osteogenic differentiation in adipose-derived mesenchymal stem cells and bone regeneration in osteoporosis via miR-33a-5p. J. Cell Mol. Med. 2020;24:12513–12524. doi: 10.1111/jcmm.15792. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 44.Zhao X., You L., Wang T., Zhang X., Li Z., Ding L., Li J., Xiao C., Han F., Li B. Enhanced osseointegration of titanium implants by surface modification with silicon-doped titania nanotubes. Int. J. Nanomed. 2020;15:8583–8594. doi: 10.2147/ijn.s270311. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 45.Miettinen S.S., Jaatinen J., Pelttari A., Lappalainen R., Mönkkönen J., Venesmaa P.K., Kröger H.P. Effect of locally administered zoledronic acid on injury-induced intramembranous bone regeneration and osseointegration of a titanium implant in rats. J. Orthop. Sci. 2009;14:431–436. doi: 10.1007/s00776-009-1352-9. [DOI] [PubMed] [Google Scholar]
  • 46.Shapiro G., Lieber R., Gazit D., Pelled G. Recent advances and future of gene therapy for bone regeneration. Curr. Osteoporos. Rep. 2018;16:504–511. doi: 10.1007/s11914-018-0459-3. [DOI] [PubMed] [Google Scholar]
  • 47.Aghali A. Craniofacial bone tissue engineering: current approaches and potential therapy. Cells. 2021;10:2993. doi: 10.3390/cells10112993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 48.Enuka Y., Lauriola M., Feldman M.E., Sas-Chen A., Ulitsky I., Yarden Y. Circular RNAs are long-lived and display only minimal early alterations in response to a growth factor. Nucleic Acids Res. 2016;44:1370–1383. doi: 10.1093/nar/gkv1367. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 49.Holdt L.M., Kohlmaier A., Teupser D. Circular RNAs as therapeutic agents and targets. Front. Physiol. 2018;9:1262. doi: 10.3389/fphys.2018.01262. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 50.Yin C., Zhang Y., Cai Q., Li B., Yang H., Wang H., Qi H., Zhou Y., Meng W. Effects of the micro-nano surface topography of titanium alloy on the biological responses of osteoblast. J. Biomed. Mater. Res. A. 2017;105:757–769. doi: 10.1002/jbm.a.35941. [DOI] [PubMed] [Google Scholar]
  • 51.Zhou R., Miao S., Xu J., Sun L., Chen Y. Circular RNA circ_0000020 promotes osteogenic differentiation to reduce osteoporosis via sponging microRNA miR-142-5p to up-regulate bone morphogenetic protein BMP2. Bioengineered. 2021;12:3824–3836. doi: 10.1080/21655979.2021.1949514. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 52.Guo Z., Zhao L., Ji S., Long T., Huang Y., Ju R., Tang W., Tian W., Long J. CircRNA-23525 regulates osteogenic differentiation of adipose-derived mesenchymal stem cells via miR-30a-3p. Cell Tissue Res. 2021;383:795–807. doi: 10.1007/s00441-020-03305-7. [DOI] [PubMed] [Google Scholar]
  • 53.Ji H., Cui X., Yang Y., Zhou X. CircRNA hsa_circ_0006215 promotes osteogenic differentiation of BMSCs and enhances osteogenesis-angiogenesis coupling by competitively binding to miR-942-5p and regulating Runx2 and VEGF. Aging (Albany NY) 2021;13:10275–10288. doi: 10.18632/aging.202791. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 54.Babij P., Zhao W., Small C., Kharode Y., Yaworsky P.J., Bouxsein M.L., Reddy P.S., Bodine P.V., Robinson J.A., Bhat B., et al. High bone mass in mice expressing a mutant LRP5 gene. J. Bone Miner Res. 2003;18:960–974. doi: 10.1359/jbmr.2003.18.6.960. [DOI] [PubMed] [Google Scholar]
  • 55.Lim W.H., Liu B., Mah S.J., Yin X., Helms J.A. Alveolar bone turnover and periodontal ligament width are controlled by Wnt. J. Periodontol. 2015;86:319–326. doi: 10.1902/jop.2014.140286. [DOI] [PubMed] [Google Scholar]
  • 56.Jiang Z., Wang H., Yu K., Feng Y., Wang Y., Huang T., Lai K., Xi Y., Yang G. Light-controlled bmsc sheet-implant complexes with improved osteogenesis via an LRP5/β-catenin/Runx2 regulatory loop. ACS Appl. Mater. Inter. 2017;9:34674–34686. doi: 10.1021/acsami.7b10184. [DOI] [PubMed] [Google Scholar]
  • 57.Trapani I., Puppo A., Auricchio A. Vector platforms for gene therapy of inherited retinopathies. Prog. Retin. Eye Res. 2014;43:108–128. doi: 10.1016/j.preteyeres.2014.08.001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 58.Siljak-Yakovlev S., Pustahija F., Vicic V., Robin O. Molecular cytogenetics (FISH and fluorochrome banding): resolving species relationships and genome organization. Methods Mol. Biol. 2014;1115:309–323. doi: 10.1007/978-1-62703-767-9_15. [DOI] [PubMed] [Google Scholar]

Associated Data

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Supplementary Materials

Document S1. Figures S1–S5 and Tables S1 and S2
mmc1.pdf (4.5MB, pdf)
Document S2. Article plus supplemental information
mmc2.pdf (8.1MB, pdf)

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