Cyclic Peptide D7 Promotes Osteogenesis and Inhibits Osteoclastogenesis by Regulating Redox Balance of Bone Remodeling


 BackgroundMilitary training usually causes excessive physical burden to the bones and joints of soldiers, which in turn disturbs the balance of bone homeostasis and eventually induces bone metabolic diseases. In our previous studies, a cyclic peptide D7 in was characterized through phage display technology from the peptide phage display library (Ph.D.‑C7C) and was proven to enhance the adhesion, expansion, and proliferation of bone marrow mesenchymal stem cells (BMSCs) on the biomaterial scaffold. However, we found that cyclic peptide D7 also has a certain affinity for mouse calvarial osteoblast precursor cells (OPCs) and bone marrow-derived monocytes/macrophages (BMMs). To investigate whether D7 can protect physiological bone remodeling and maintain bone homeostasis, we elucidated the mechanisms by which D7 promotes osteogenesis and inhibits osteoclastogenesis. MethodsThe affinity of D7 peptide towards calvarial OPCs and BMMs was investigated using fluorescence cytochemistry. The roles of D7 in osteogenesis and osteoclastogenesis by regulating redox balance was confirmed by cell counting kit-8, special stain assays (alkaline phosphatase stain, alizarin red stain and tartrate‐resistant alkaline phosphatase stain), fluorescence cytochemistry, western blotting, quantitative real-time polymerase chain reaction. ResultsThe results demonstrated that D7 promoted the osteogenesis of calvarial OPCs by upregulating the expression of osteogenic differentiation factors. In contrast, D7 inhibited osteoclast differentiation and resorption by downregulating the expression of osteoclast phenotype marker. We further identified that these phenomenons may be due to the suppressive effect of D7 on reactive oxygen species production and the enhancing effect on antioxidant expression in both OPCs and BMMs, thereby regulating the redox balance of bone remodeling.Conclusions﻿Cyclic peptide D7 could maintain bone homeostasis by regulating redox balance and provide a potential approach to strengthen the skeletal fitness of soldiers and prevent the occurrence of bone metabolic diseases.

The interaction between bone formation caused by osteoblasts and bone resorption caused by osteoclasts is tightly regulated during the bone remodeling process [6]. Osteogenic proteins are produced by osteoblasts, such as alkaline phosphatase (ALP) and type I collagen (COL1). unmineralized extracellular matrix is rst secreted in a bone-like form, and then mineralized to form new bone through the accumulation of calcium phosphate [7], which can secrete acid and proteolytic enzymes, such as cathepsin K (CTSK), and degrade bone by dissolving collagen and other matrix proteins [8]. Interestingly, there is a connection between osteoblasts and osteoclasts. They can communicate with each other through direct contact, such as the bidirectional signal transduction effect of ephrin B2-ephrin B4 [9], to regulate the differentiation and survival of osteoblasts and osteoclasts. Under the in uence of estrogen, osteoblasts can also regulate the Fas-Fas ligand pathway through paracrine action to regulate the function and status of pre-osteoclasts [10,11]. In addition, osteoblasts can also produce a series of secreted molecules, including macrophage colony-stimulating factor (M-CSF) [12,13] and receptor activator of nuclear factor-κB ligand (RANKL)/osteoprotegerin [14,15], which promote or inhibit the proliferation, differentiation, and migration of osteoclasts. Osteoclasts can also secrete soluble factors that affect the survival, migration, and differentiation of osteoblasts. These soluble factors include sphingosine 1-phosphate [16], collagen triple helix repeat-containing 1 [17], and complement component 3 [18]. In addition, many bone diseases are associated with oxidative stress. Oxidative stress changes the bone remodeling process, leading to an imbalance in the activity of osteoclasts and osteoblasts, which may lead to metabolic bone diseases, such as osteoporosis [19,20]. Reactive oxygen species (ROS) induce osteoblast and osteocyte apoptosis [21,22], which is conducive to osteoclast generation and inhibits mineralization and osteogenesis. Antioxidants activate the mineralization of osteoblasts and promote their osteogenic differentiation and decrease the activity of osteoclasts through confront oxidants [22,23,24]. Therefore, a drug is needed that promotes osteogenesis and inhibits osteoclasts, while maintaining redox balance.
In our previous experiments, the cyclic 7 peptide (CDNVAQSVC), named D7, was discovered using phage display technology from the peptide phage display library (Ph.D.-C7C). We used the different binding a nities between the cycle peptide and the target cells described in a previously published protocol to obtain D7 through three biopanning procedures [25]. Moreover, we showed that a modi ed D7 scaffold enhances the adhesion, expansion, and proliferation of bone marrow mesenchymal stem cells (BMSCs) [26], but its mechanism of action is still unclear. In the present study, we investigated the ability of D7 to affect osteogenesis and osteoclastogenesis via the regulation of intracellular redox, providing evidence for D7 as a potential drug candidate to prevent pathological fracture and bone metabolism diseases.
When the cells reached 80% con uency, osteoinduction medium (OIM; containing 100 nM dexamethasone, 10 mM β-glycerophosphate disodium salt hydrate, and 50 ug/ml ascorbic acid on the basis of CM) was added. The induction media were replaced every two days.
Bone marrow-derived monocytes/macrophages (BMMs) were ushed from the femurs and tibiae of 8week-old C57BL/6 male mice using α-MEM. After centrifugation for 10 min at 1500 rpm, α-MEM containing 10% FBS and 1% P/S was added, and the mixture was incubated at 37°C for 1 d. Nonadherent cells were harvested and centrifuged for 10 min at 1500 rpm. After addition of red blood cell lysate for 3 min and centrifugation for 5 min at 1500 rpm, 2×10 5 /ml cells were cultured in α-MEM supplemented with 10% FBS, 1% P/S, and 25 ng/ml M-CSF for 3 days. Adherent cells were used as osteoclast precursors. Then, osteoclast precursors were incubated with 40 ng/ml RANKL in α-MEM containing 10% FBS, 1% P/S, and 25 ng/ml M-CSF for 5 days. The culture medium was refreshed on days 3, 4, and 5.

ALP staining and Alizarin Red staining
Calvarial OPCs were treated with or without 20 µM D7 in OIM for 5 days. Calvarial OPCs were xed in 4% paraformaldehyde for 20 min, incubated with ALP substrate solution according to the manufacturer's instructions at 37°C in the dark for 30 min, and then visualized under a microscope (IX53; Olympus, USA).
After incubation with or without 20 µM D7 in OIM for 21 days, the cells were xed in 4% paraformaldehyde for 20 min, washed with PBS three times, and stained with 1% Alizarin Red (pH 4.1) for 10 min. Alizarin staining was determined using colorimetric analysis after washing three times with PBS.

TRAP and bone resorption assay
After osteoclast differentiation in 24-well plates, the cells were xed with 4% paraformaldehyde for 20 min and stained using TRAP according to the manufacturer's instructions. TRAP-positive multinucleated cells containing three or more nuclei were counted as osteoclasts under a light microscope (IX53; Olympus, USA).
After mature osteoclasts were formed in 96-well Osteo Assay Surface plates (cat. no. 3988; Corning, USA), which were coated with a hydroxyapatite matrix, at a density of 1×10 4 cells /well, the medium was aspirated completely, and 10% bleach solution was added to each well for 5 min to remove the cells. The wells were then washed twice with deionized water, and the plates were air-dried before evaluation.
Absorption pits were observed using a light microscope (IX53; Olympus, USA).

Actin-ring staining
After mature osteoclasts were formed in 24-well plates, cells were xed with 4% paraformaldehyde for 20 min, permeabilized with 0.1% Triton X-100, and incubated with Phalloidin-iFluor 594 (cat. no. ab176757; Abcam, USA) for 45 min at room temperature. The cells were then incubated with 10 µg/ml DAPI (Beijing Solarbio Science and Technology, Inc.) for 5 min at room temperature. The plates were imaged under a high-content microscope (ImageXpress Micro Confocal; Molecular Devices, USA).
Acridine Orange (AO) staining AO (cat. no. A6014; Sigma-Aldrich, USA) stock solution (1 mg/ml) was prepared in α-MEM. After mature osteoclasts were formed in 24-well plates, the medium was aspirated, and osteoclasts were incubated with AO solution (10 µg/ml) for 15 min at 37°C. Cells were washed twice with α-MEM and observed under a Nikon uorescence microscope (Axio Observer 3; Carl Zeiss AG, Germany). The quantitative experiment was performed in the same way in 96-well plates. The absorbance of the cells was measured at 458 nm using a spectrophotometer (Multiskan GO 1510; Thermo Fisher Scienti c, USA).

WB analysis
Brie y, proteins were extracted from each sample using a radioimmunoprecipitation assay buffer (cat. no. R0020; Solarbio, China) containing a protease inhibitor (cat. no. CW2200; Cwbio, China) and phosphatase inhibitors (cat. no. CW2383; Cwbio, China) and quanti ed using a bicinchoninic acid protein assay kit (cat. no. PC0020; Solarbio, China). Then, 20 µg of protein from each sample was separated using 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred onto a 0.22 µm polyvinylidene di uoride membrane (cat. no. IPVH00010; Millipore, Billerica, MA, USA) for 140 min at 180 mA. After blocking with 5% skim milk in Tris-buffered saline containing 0.1% Tween 20 (TBST) for 1 h at room temperature, the membranes were incubated with the primary antibodies (NFATc1, TRAF6, CTSK, RUNX2, ALP, COL1a1, and HRP-β-actin) overnight at 4°C. After washing in TBST three times (8 min each time), the membranes were incubated with the respective secondary antibodies at room temperature for 1 h. Chemiluminescence was measured using an Immobilon Western Chemiluminescent HRP Substrate (cat. no. wbkls0500; Millipore, USA). Band densities were quanti ed using the ImageJ software.
Quantitative real-time polymerase chain reaction (RT-qPCR) Total ribonucleic acid RNA was isolated using RNAiso Plus (cat. no. 9109; Takara, Tokyo, Japan). After dissolving the extracted RNA in RNase-free distilled water, used spectrophotometry to determine the quality and quantity of the RNA samples. The absorbance ratio at 260nm and 280nm is maintained between 1.8 and 2.1. Next, according to the manufacturer's instructions, total RNA was reversetranscribed into complementary deoxyribonucleic acid (DNA) using a PrimeScript™ RT Reagent Kit with a genomic DNA Eraser (cat. no. RR047A; Takara). Speci c transcripts were quanti ed by RT-qPCR using the QuantiTect SYBR Green PCR Kit (cat. no. AG11701; Accurate Biology, China) and analyzed using a RT-PCR system. Gene-speci c primers used for RUNX2, ALP, COL1, NFATc1, CTSK, TRAF6, Cu/Zn superoxide dismutase (SOD), Mn SOD, Catalase (CAT), glutathione reductase (GR), glutathione peroxidase (GPX), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), and β-actin are listed in Addition table 1. The messenger RNA (mRNA) levels of RUNX2, ALP, COL1, NFATc1, CTSK, and TRAF6 were normalized to βactin mRNA levels. The mRNA levels of Cu/Zn-SOD, Mn-SOD, CAT, GR, and GPX were normalized to GAPDH mRNA levels. PCR was performed at 95°C for 5 seconds, 40 cycles were performed, and 65°C for 30 s. The comparative CT method was used to calculate relative mRNA expression.

Intracellular ROS measurement
Intracellular ROS formation was detected using a DCFH-DA probe (cat. no. s0033; Beyotime, China). Calvarial OPCs and BMMs were cultured in 6-well plates. Calvarial OPCs were treated with or without 20 µM D7 in OIM for 24 h, and BMMs were treated with or without 20 µM D7 in M-CSF (25 ng/ml) and RANKL (40 ng/ml) for 24 h. Cells were then washed using α-MEM and loaded with 10 µM DCFH-DA in serum-free α-MEM at 37°C for 20 min and then washed three times using α-MEM. Fluorescence intensity was determined using a Nikon uorescence microscope (Axio Observer 3; Carl Zeiss AG, Germany).

Statistical Analysis
Data are presented as mean ± SD and statistical analyses were performed using unpaired Student's T-Test. Differences among groups were assessed using one-way analysis of variance (ANOVA). All data analyses were conducted using Graphpad software. P value of < 0.05 was considered to be statistically signi cant.

D7 maintained bioactivity in the cytoplasm and had no cytotoxicity to calvarial OPCs and BMMs
To verify the a nity of D7 for calvarial OPCs and BMMs, the calvarial OPCs of C57BL/6 mice were incubated with 20 µM FITC-labeled D7 for 48 h. Fluorescence microscopy revealed that FITC uorescence was concentrated in the cytoplasm of calvarial OPCs, but no obvious FITC uorescence signal appeared in the nucleus. Similarly, FITC uorescence was also concentrated in the cytoplasm under the same conditions in mouse BMMs, and no obvious FITC uorescence signal was observed in the nucleus ( Figure   1B). The results show that D7 has a certain a nity for calvarial OPCs and BMMs, and that it can enter cells to play a certain role.
To evaluate the toxicity of D7 to these two kinds of cells. Calvarial OPCs were cultured with different concentrations of D7 in CM and OIM for 48 h. The results in CM showed that the activity of osteoblast precursor cells increased slightly with the increase in D7 concentration, and there was no toxicity within 200 µM ( Figure 1C). In the OIM, when the concentration of D7 was 20-200 µM, the proliferation of calvarial OPCs was slightly inhibited ( Figure 1D). Similarly, different concentrations of D7 and BMMs were co-cultured for 48 h to determine toxicity. As the concentration of D7 increased, the toxicity to BMMs increased slightly, and D7 was signi cantly toxic to BMMs at a concentration of 100 µM ( Figure 1E). With the increase in D7 concentration, the cell viability of BMMs induced by RANKL showed a slight increase ( Figure 1E). The toxicity assay suggested that D7 is not cytotoxic to calvarial OPCs and BMMs at low and medium doses.

D7 promotes osteoblast differentiation
To analyze the role of D7 in promoting osteogenic differentiation of calvarial OPCs, the cells were induced in OIM with or without D7 for 5 days for ALP staining, and 21 days for induction of Alizarin Red staining. Compared with the OIM alone group, D7 promoted the formation of ALP and increased the formation of mineralized nodules (Figure 2A). Next, we analyzed the gene expression levels of the osteogenic differentiation markers ALP, RUNX2, and COL1a1 in calvarial OPCs treated with 20 µM D7 by RT-qPCR and WB to determine the mechanism of D7 action on osteoblast differentiation. Compared with the OIM alone group, the expression of ALP, RUNX2, and COL1a1 was signi cantly upregulated by D7 at both mRNA ( Figure 2B, C, D) and protein levels ( Figure 2E, F, G, H). These results demonstrated that 20 µM D7 promotes the osteogenic differentiation of calvarial OPCs.

D7 inhibits RANKL-induced osteoclast differentiation
To better understand the effect of D7 on RANKL-induced osteoclast differentiation, BMMs were induced in RANKL with or without D7 for 5 days for TRAP staining. Compared with the control group, D7 (20 µM) effectively inhibited the fusion of osteoclasts induced by RANKL, thereby inhibiting osteoclast differentiation ( Figure 3A). We next investigated the mechanism of action of D7 on osteoclast differentiation using RT-qPCR and WB. The expression of osteoclast markers NFATc1, TRAF6, and CTSK increased at both mRNA ( Figure 3B, C, D) and protein levels ( Figure 3E, F, G, H). These results show that 20 µM D7 can reverse the maturation and differentiation of osteoclasts induced by RANKL by inhibiting the expression of these bone resorption-related enzymes.

D7 inhibits RANKL-induced osteoclast function
To further understand the in uence on osteoclast function by D7, mature osteoclasts resulted in resorption pits through calcium absorption, and D7 reduced the area of the resorption pits ( Figure 4A). Due to the stimulation of RANKL, the formation of the F-actin rings increased. However, D7 notably suppressed the number and size of the structure of F-actin rings ( Figure 4B). AO binds to RNA in the cytoplasm and emits green uorescence; the levels of AO in acidic vesicles were high, and all stained acidic vesicles emit red uorescence. D7 reduced the formation and release of acidic vesicles with osteolytic activity ( Figure 4C). These results con rmed that 20 µM D7 suppressed RANKL-induced reduction of osteoclast function.

D7 maintains the redox balance of calvarial OPCs and BMMs
To verify whether D7 promotion of osteogenic differentiation and inhibition of osteoclast differentiation are caused by the inhibition of ROS production, intracellular ROS were detected in both calvarial OPCs and BMMs. D7 reduced the formation of ROS in calvarial OPCs during osteogenic differentiation ( Figure   5A). Whereas RANKL-induced ROS formation was reduced in BMMs after treatment with D7 for 24 h ( Figure 5B). We next assessed the levels of antioxidative-associated substances Cu/Zn SOD, Mn SOD, CAT, GPX, and GR by RT-qPCR. After treatment with D7, the expression of these substances increased in the calvarial OPCs, compared with that in the calvarial OPCs in the OIM alone group ( Figure 5C, D, E, F, G). Compared with the control group, the levels of these antioxidative-associated substances remained same ( Figure 5H, I, J, K, L), showing that 20 µM of D7 maintained the redox balance both in calvarial OPCs and BMMs.

Discussion
Polypeptides have gradually entered the public eld of vision because of their advantages in medical research. They are characterized by high biological activity, strong speci city, benign amino acids, low toxicity, and ease of synthesis and modi cation [27]. To date, a large number of studies have proven the effect of polypeptides in the treatment of bone diseases in many ways, such as improving cell adhesion to speci c biomaterial surfaces, promoting osteogenic differentiation [28], and decreasing infection rate, thereby causing less in ammatory damage [29]. On this basis, cyclic peptides provide higher a nity [30,31], better stability [32] and better osteointegration [33] than linear peptides because of their cyclic structure. Our previous study used the Ph.D.C7C™ phage display library to screen the cyclic peptide D7, and con rmed that it enhanced the adhesion, expansion, and proliferation of BMSCs on the biomaterial scaffold. In the present study, we con rmed that cyclic peptide D7 not only promotes bone formation, but also inhibits osteoclasts by regulating the redox balance of bone remodeling. Therefore, D7 may become an important drug candidate for strengthening the bones of soldiers to prevent pathological fractures or bone metabolism diseases by regulating bone remodeling.
Cyclic peptides are known to protect cells from oxidative stress damage [34], and some peptides were found to exhibit anti-in ammatory properties [35]. A redox imbalance causes oxidative stress, as the activity of ROS-producing enzymes and antioxidants cannot maintain this balance [36]. Redox imbalance changes the bone remodeling process. It can activate the differentiation of preosteoclasts and strengthen bone resorption [37]. In the present study, the formation of intracellular ROS was signi cantly decreased in both two cell types. However, the reduction of intracellular ROS leads to osteogenesis enhancement and weakened osteoclastogenesis and bone resorption. It can be seen that ROS induce osteoblast apoptosis, which is bene cial to the generation of osteoclasts [38]. In addition, intracellular ROS must be degraded by antioxidants, such as Cu/Zn SOD, Mn SOD, CAT, GPX, and GR [39]. D7 balances the production of intracellular ROS by increasing the level of antioxidant enzymes, which balances the levels of ROS and antioxidants in the cell to protect it from ROS damage. Therefore, treatment with D7 regulated the redox balance and maintained the balance between osteoclast and osteoblast activity, in turn regulating bone remodeling.
The regulation of bone remodeling is controlled by a dynamic balance between osteoblast bone formation and osteoclast bone resorption. Therefore, in the present study, we used two cell models to con rm the effects of cyclic peptide D7, mouse calvarial OPCs, and BMMs.
Calvarial OPCs are derived from mouse skulls, which are helpful for studying the differentiation and proliferation of bone cells [40]. Mineralized nodules are biomarkers that determine the maturation of osteoblasts, which can be detected by Alizarin Red staining [41]. In the present study, more mineralized nodules were formed after treatment with D7. During the differentiation of osteoblast precursor cells into mature osteoblasts, the expression of RUNX2 increases [42]. ALP expression increases during early osteogenic differentiation [43]. COL1a1 is also a speci c marker of osteogenesis, whose expression is upregulated in the late stage of osteogenic differentiation [44]. Consistent with these data, we observed that D7 induced the upregulation of the expression of these three speci c markers of osteogenesis at the gene and protein levels. These results clearly indicate that D7 promoted osteogenesis. Most importantly, the cyclic peptide was not toxic to calvarial OPCs at a concentration of 200 µM. Under osteogenic induction, cell proliferation decreased slightly with increasing D7 concentration, which may be due to calvarial OPC preference for osteogenic differentiation compared to continued proliferation.
Mononuclear macrophages are derived from mouse bone marrow. They act as pre-osteoclasts induced by RANKL and differentiate into mature osteoclasts to induce bone resorption, and they are used to establish an experimental model of RANKL-induced osteoclasts [45]. TRAP is an osteoclast phenotype marker that also affects the differentiation and maturation of osteoclasts. TRAP staining is a standard method for the detection of osteoclast differentiation [46]. In the present study, D7 decreased the number of mature osteoclasts induced by RANKL. At the same time, the function of mature osteoclasts was inhibited by D7 treatment. Mature osteoclasts have a closed area composed of F-actin rings, which form the most visible feature of mature osteoclasts [47]. Acidic vesicles are secreted by mature osteoclasts, which play a role in bone resorption and can be imaged and tracked in cells after staining with AO [48]. The bone resorption assay was used to determine the bone resorption capacity of mature osteoclasts [47]. In addition, we con rmed that D7 reduces the expression of early osteoclast markers NFATc1 and TRAF6 at the gene and protein levels. NFATc1 is a key determinant of osteoclastogenesis [6]. CTSK, an essential enzyme released when osteoclasts undergo bone resorption [49] is regulated by NFATc1 [50], and the expression of CTSK was reduced by D7 compared with the control group. These results con rm that D7 inhibits the differentiation and function of osteoclasts induced by RANKL by signi cantly suppressing bone resorption-related factors.

Conclusions
In conclusion, we demonstrated that D7 promotes osteogenesis and inhibits osteoclastogenesis by maintaining redox balance during bone remodeling. D7 may be able to improve injuries to the bones and joints of soldiers due to long-term military training and prevent the occurrence of bone metabolic diseases, such as osteoporosis.    presented as the means ± standard error of the mean (n=3). ***p < 0.001 versus control group. (C) Cells were stained with AO and observed under a uorescence microscope. Scale bar = 100 μm. The results are presented as the means ± standard error of the mean (n=3). *p<0.05 versus control group. BMMs, bone marrow-derived monocytes; D7, CDNVAQSVC; DAPI, 4′,6-diamidino-2-phenylindole; M-CSF, macrophage colony-stimulating factor; RANKL, receptor activator of nuclear factor-κB ligand; AO, Acridine Orange.