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Osthole is a bioactive coumarin derivative and has been reported to be able to enhance bone formation and improve fracture healing. However, the molecular mechanism of Osthole in bone fracture healing has not been fully defined. In this study we determined if Osthole enhances bone fracture healing through activation of BMP2 signaling in mice. We performed unilateral open transverse tibial fracture procedure in 10-week-old C57BL/6 mice which were treated with or without Osthole. Our previous studies demonstrated that chondrocyte BMP signaling is required for bone fracture healing, in this study we also performed tibial fracture procedure in Cre-negative and Col2-Cre;Bmp2flox/flox conditional knockout (KO) mice (Bmp2Col2Cre) to determine if Osthole enhances fracture healing in a BMP2-dependent manner. Fracture callus tissues were collected and analyzed by X-ray, micro-CT (μCT), histology, histomorphometry, immunohistochemistry (IHC), biomechanical testing and quantitative gene expression analysis. In addition, mouse chondrogenic ATDC5 cells were cultured with or without Osthole and the expression levels of chondrogenic marker genes were examined. The results demonstrated that Osthole promotes bone fracture healing in wild-type (WT) or Cre- control mice. In contrast, Osthole failed to promote bone fracture healing in Bmp2Col2Creconditional KO mice. In the mice receiving Osthole treatment, expression of cartilage marker genes was significantly increased. We conclude that Osthole could promote bone strength and enhance fracture healing by activation of BMP2 signaling. Osthole may be used as an alternative approach in the orthopaedic clinic for the treatment of fracture healing.
Bone fractures are one of the most common traumatic injuries in humans 1, especially among elders with advanced osteoporosis 2. The time length of the fracture healing is an important factor in determining a patient's recovery rate and treatment cost 3. Bone fracture heals normally in most patients; however, an overall 5 to 10% of bone fractures result in hypertrophic callus formation and delayed healing process 4.
Clinically, the fracture healing process is determined by patients' health conditions, anatomic locations, as well as quality of the surrounding soft tissues. Temporary immobilization and surgical fixation are considered as the treatment choice for most fractures 5. Unfortunately, many patients refused to undergo invasive operation due to the risk of non-union and delayed union 6. There is a critical need to develop alternative strategies to promote fracture healing and repair.
Osthole (7-methoxy-8-isopentenoxycoumarin, C15H16O3, 244.39 Da) is a bioactive coumarin derivative 7-9 and has been used clinically in the treatment of kidney diseases 10. Recently, several reports demonstrate that Osthole could prevent bone loss and promoting bone formation 11-13.
Fracture healing process is a specialized repair process that recapitulates aspects of embryonic skeletal development 14, 15. During the healing process, both periosteal and endochondral ossification processes are involved in new bone formation and the latter process plays a more important role during fracture healing. In addition, multiple growth factor signaling pathways may be activated during fracture healing process 16, 17. Among them, bone morphogenetic proteins (BMPs) play a more important role in fracture healing process. Recent studies suggest that BMP pathway is associated with the early phase in Osthole-induced osteoblast differentiation 18, 19. In addition, Osthole induces endochondral ossification by up-regulation of mature osteoblast marker genes 20. However, the underlying mechanism of Osthole-induced chondrocyte differentiation during fracture healing was not well characterized. Our current study aimed to investigate the in vivo effects and the molecular mechanisms by which Osthole promotes bone fracture healing.
Osthole (No. 110822-200305, purity > 98%, 244.39 Da) (Figure (Figure1A)1A) was purchased from the National Institute for the Control of Pharmaceutical and Biological products (Beijing, China) and was dissolved in dimethylsulfoxide (DMSO) (less than 0.1%).
10-week-old male C57BL/6 mice were obtained from the animal center of the Zhejiang Chinese Medical University (Y2111184). They were divided into two groups: Osthole treatment group (30 mg/kg, local subcutaneous injection, daily for 28 days) 11 and vehicle control (PBS) group.
To generate chondrocyte specific Bmp2 conditional KO mice, Bmp2flox/flox mice (obtained from Dr. Di Chen, Rush University, Chicago, USA) 21, 22 were bred with Col2-Cre transgenic mice. The Col2-Cre transgenic mice could efficiently target growth plate chondrocytes 23, 24. All mice were under C57BL/6 background and mouse genotyping was determined by PCR using DNA extracted from tail biopsy tissues. PCR primer sequences for genotyping were as follows, MP1-600 F (A) 5'-AGGGTTTCAGGTCAGTTTCCG, MP1-Hap-rv2 (B) 5'-GATGATGAGGTTCTTGGCGG, and MP1-1615RW (C) 5'-TCCGAAGGTAAGTGTGCTTGG. Cre-positive and Bmp2flox/flox male littermates were chosen as experimental groups while Cre-negative male littermates were used as controls (mean body weight 22 ± 2g). All mice had free access to food and water during the entire study.
In this study, a unilateral (right side) open transverse tibial fracture with intramedullary needle fixation was selected as the bone fracture model in 10-week-old C57BL/6, Cre-negative and Bmp2Col2Cre mice 22, 25, 26. Anesthesia was induced with Ketamine (60 mg/Kg) via intraperitoneal injection. Next a 1.5cm-long incision was made along the anterior-medial surface and a 26 gauge syringe needle was inserted into the bone marrow cavity of the tibia through the tibial plateau on the medial side of the patellar ligament. Then the needle was removed, and a No. 11 surgical blade was used to transect the diaphysis of the tibia at the midpoint. A 26 gauge needle was then inserted into the tibia to stabilize the fracture. A 4-0 silk suture was used to close the wound and pain was managed using buprenorphine (in drinking water) for the first three days after surgery 26.
After operation, X-ray radiographic analysis was performed immediately after fracture to ensure the fracture pattern and the position of the fixation needle. Mice were sacrificed at days 7, 10, 14, 21 and 28 for histological analysis after surgery (n=10 in each time point). Fracture healing was examined by the assessment of bridging across cortices. The extent of bridging between the fracture gap was determined in a blinded pattern by three independent investigators following the criteria: 1) grade 1: no healing (no indication or only some indications mineralized bridging); 2) grade 2: partial healing (more consolidated bridging or almost completely mineralized bridging); and 3) grade 3: complete healing (completely mineralized bridging). Specimens were scanned at 10.5-micron isotropic resolution using a micro-computed tomography (μCT) (Skyscan 1176; Bruker μCT, Kontich, Belgium) at each time points 26, 27. Callus total volume (TV), callus bone volume (BV), callus mineralized volume fraction (BV/TV) (%) and callus bone mineral density (BMD) were determined (n=10 in each time point).
Tibia bone samples were harvested and surrounding soft tissues were carefully removed (n=10 at days 10, 14, 21, and 28). Samples were fixed in aluminum square tubes (0.5 cm) filled with bone cement to make sure that fracture lines were in the middle of the interval. Specimens were mounted on an EnduraTec TestBenchTM system with a 200 N.mm torque cell (EnduraTec TestBenchTM system, Bose Corp., Minnetonka, MN) and tested in torsion at a rate of 10/sec until failure to determine maximum torque, stiffness and toughness of fracture callus 22.
Bone samples were harvested (n=10 at days 7, 10, 14, 21 and 28) and fixed in 10% normal buffered formaldehyde for 3 days, decalcified in 14% EDTA solution for 14 days at room temperature and then embedded in paraffin. Tissue sections at the fracture site were cut longitudinally at thickness of 3-μm and prepared for Alcian blue/H&E staining and TRAP staining (day 14). Histomorphometric analysis (n=10 in each time point) was conducted using OsteoMetrics software (Decatur, GA). The mineralized volume of the cortices, the area of the periosteal calluses, and the mineralized and cartilaginous volume of the calluses were measured 22, 26. The numbers of TRAP-positive multinucleated osteoclasts and percentage of osteoclast surface of the calluses were also measured. IHC was performed using anti-type II collagen (NeoMarkers, Inc. Fremont, CA, USA) and anti-pSmad1/5/8 (Santa Cruz Biotechnology, Santa Cruz, CA, USA) antibodies on the 3-μm thick tissue sections (days 7).
Total RNA was extracted from the callus tissue including adjacent bone on either side of the fracture line (1 mm) using the PureLink™ RNA Mini Kit (Invitrogen, Carlsbad, CA, USA) (n=6 in each time point). One microgram total RNA was used to synthesize cDNA using iScripts cDNA Synthesis Kit (Bio-Rad, Hercules, CA, USA). Real-time PCR amplification was performed using murine gene specific primers and SYBR green real-time PCR kit (Bio-Rad, Hercules, CA, USA). The levels of the target gene expression were normalized to that of β-actin. Expression levels of chondrogenic (Sox9, Col2a1, Col10a1) and osteogenic (Runx2, osteocalcin) marker genes was examined. Forward and reverse primers specific for the genes listed in Table Table11.
Mouse chondrogenic ATDC5 cells (RIKEN Cell Bank, Tsukuba, Japan) were cultured in maintenance medium containing 1:1 Dulbecco's modified Eagle medium (DMEM)/F-12 medium (Sigma, St. Louis, MO, USA), supplemented with 5% fetal bovine serum (FBS), 100 U/mL penicillin,100 mg/mL streptomycin (Gibco, Grand Island, NY, USA), 1% L-glutamine (Gibco). After reaching 100% confluent, cells were centrifuged and then were plated in 12-well plates (Corning, NY, USA) at the center of each well for 2-3 h before adding chondrogenic differentiation medium (maintenance medium plus 10 μg/mL insulin, 10 μg/ml transferrin and 30 nM sodium selenite). All cells were maintained at 37°C in a humidified 5% CO2 incubator. The medium was changed every other day. After 2 days of culture, cells were treated with Osthole at concentrations of 0, 10, 50, 100 µM for one week (6 wells per group) 11. Then total RNA was extracted and the levels of chondrogenic associated genes (Sox9, Col2a1, Col10) were analyzed.
All values were presented as mean ± standard deviation. Statistical analyses included unpaired Student's t-tests and one-way ANOVA test followed by the Tukey-Kramer test. The statistical tests were performed using the software SPSS 17.0. P < 0.05 was considered as significant.
Osthole (7-methoxy-8-isopentenoxycoumarin, C15H16O3, 244.39 Da) (Figure (Figure1A)1A) is an O-methylated coumarin isolated from plants such as Cnidium monnieri, Angelica archangelica and Angelica pubescens 29. In the present studies, we examined the effect of Osthole on bone fracture healing. The results of radiographic analysis showed that administration of Osthole enhanced bone callus formation. Compared to the PBS control group, the presence of a fracture gap was blurry in Osthole treated group at days 10, 14 and 21 (Figure (Figure1B1B and Table Table22).
Moreover, there was almost no fracture gap visible at day 28 in Osthole treated group, thus further confirming the beneficial role of Osthole on the normal fracture healing process (Figure (Figure1B).1B). In contrast the control group showed obvious fracture gap at the same time point. μCT data showed that the bone volume in Osthole-treated mice was increased compared to PBS control group at days 7, 10 and 14 (Figure (Figure2A-C),2A-C), supporting the radiographic findings. Results of biomechanical testing showed that treatment with Osthole enhanced bone mechanical strength (Figure (Figure2D-F).2D-F). The maximum torque and stiffness were significantly increased at days 10 and 14 in Osthole treated group compared to controls (Figure (Figure2D2D and E).
Histologic and histomorphometric analyses showed that the progression of fracture healing was accelerated in Osthole-treated group. Cartilage area was determined by Alcian blue staining, which is significantly enlarged by day 7 in Osthole-treated mice (Figure (Figure3A3A and B). The woven bone area was increased at days 7, 10 and 14 after surgery in Osthole-treated group (Figure (Figure3A3A and C). The increase in cartilage area was supported by gene expression results showing that expression of the early cartilage marker genes such as Sox9 and Col2a1 were increased at day 7 in Osthole-treated group (Figure (Figure4A4A and B). The levels of the later cartilage hypertrophic marker Col10a1 were also increased at day 10 in Osthole-treated mice (Figure (Figure4C).4C). The expression of bone marker genes, Runx2 and osteocalcin (OCN), was increased at day 21 in Osthole-treated group (Figure (Figure4D4D and E). The changes in expression of cartilage and bone marker genes are consistent with the process and timing of endochondral ossification during the fracture healing process.
Our findings suggest that Osthole can promote bone fracture healing by accelerating endochondral ossification. The expression of the type II collagen protein was further confirmed by IHC staining of the callus at day 7, with increased Col-II expression in Osthole-treated group (Figure (Figure5A).5A). Moreover, increased pSmad1/5/8 expression was detected at day 7 in Osthole-treated group (Figure (Figure5B).5B). Furthermore a mouse chondrogenic ATDC5 cells were used for in vitro experiments. Osthole promoted chondrogenesis in a dose-dependent manner (10-100 μM), as shown by the up-regulation of chondrocyte related marker genes such as Sox9, Col2a1 and Col10a1 (Figure (Figure55C).
To determine if Osthole regulates osteoclast formation, we also performed TRAP staining in fracture callus at day 14. No significant changes in osteoclast numbers were found after Osthole treatment (Figure (Figure6A-C),6A-C), suggesting that Osthole has no significant effect on osteoclast formation during fracture healing.
To provide the definitive evidence about the role of Bmp2 in Osthole-induced fracture healing, we examined the effect of Osthole on fracture healing in chondrocyte-specific Bmp2 conditional KO mice. Radiographic data demonstrated that treatment with Osthole enhanced bone callus formation in Cre-negative group compared to the Bmp2Col2Cre group. The appearance of a fracture gap was blurry in Cre-negative group at earlier days 10, 14 and 21 than Bmp2Col2Cregroup (Figure (Figure77 and Table Table3).3). At day 28, there was no obvious fracture gap in Cre-negative group. In contrast, obvious fracture gaps were seen in Bmp2Col2Cre group. These results indicate the essential role of BMP2 in Osthole-induced bone fracture healing process. μCT analysis demonstrated that the bone volume in Cre-negative control group was increased in response to Osthole treatment compared to Bmp2Col2Cregroup at days 7, 10 and 14 (Figure (Figure8A-C).8A-C). In addition, the mechanical strength of the tibia was significantly decreased in Bmp2Col2Cre group, compared to the Cre-negative group in the presence of Osthole (P < 0.05) (Figure (Figure8D).8D). The stiffness also significantly decreased at days 10 and 14 in Bmp2Col2Cre group, compared to Cre-negative control group in the presence of Osthole (Figure (Figure8E).8E). However, no significant effect was found on the stiffness and toughness at day 21 and 28 after Osthole treatment (Figure (Figure8E,8E, F).
Alcian blue/H&E stained sections from Cre-negative group and Bmp2Col2Cre group, both treated with Osthole, at day 7, 10, 14, 21 and 28, highlighted the characteristic differences in cartilage area of callus tissues and woven bone areas. Results of histologic and histomorphometric analyses revealed that fracture healing progress was delayed in Bmp2Col2Cregroup. At day 7, cartilage area was significantly enlarged in Cre- control group after Osthole treatment (Figure (Figure9A9A and B). And woven bone area was increased at day 10 after surgery in Cre- control group (Figure (Figure9A9A and C). To further determine the role of BMP2 in Osthole-enhanced bone fracture healing, we analyzed the gene expression as well. Our results revealed that treatment with Osthole resulted in an increase in the expression of the early cartilage marker genes, such as Sox9 and Col2a1, at day 7 and the later cartilage hypertrophic marker Col10a1 at day 10 in Cre- control mice (Figure (Figure10A-C).10A-C). The expression of bone marker Runx2 was initially increased at day 10 in Cre- control group (Figure (Figure10D).10D). In addition, Osthole did not affect the expression of bone marker OCN (Figure (Figure10E).10E). These results suggest that Osthole promotes bone fracture healing mainly through activation of BMP signaling during cartilage formation in callus tissue.
Osthole is a coumarin derivative that is present in many plants. These plants such as Cnidium monnieri and Angelica pubescens have been used as tonics and for the treatment of bone-related diseases 13, 30. Osthole stimulates bone formation both in vitro and in vivo 11, 12. In the present study, we demonstrated that Osthole promotes bone fracture healing and its effect on fracture healing could be inhibited when Bmp2 was specifically deleted in chondrocytes. The results of the current study have identified the potential utility of Osthole in acceleration of fracture healing. The mechanism of Osthole-induced fracture healing may be partially related its role in activation of BMP signaling in chondrocytes.
Fracture healing can be temporally divided into an inflammatory phase, a soft callus phase, a hard callus phase, and a remodeling phase, and these stages show some overlap 31. The bone repair process involves the coordination of multiple cell types to recover normal bone shape and function. Our data reveal that treatment with Osthole significantly accelerates the endochondral ossification in a fracture callus. Radiographic analysis showed that bridging of the fracture gap occurred at day 10 in Osthole-treated WT and Cre- control mice, which was evidently earlier than that observed in Bmp2Col2Cremice. Bone volume was significantly increased in Osthole-treated group, compared to the vehicle control group and Bmp2Col2CreKO group at days 7, 10 and 14. These data are consistent with histologic and histomorphometric analyse, showing that Osthole enhances fracture healing at the early chondrogenesis phase. Osthole-treated mice exhibited a fracture callus with a larger cartilaginous compartment compared to control group and Bmp2Col2Cre KO group at day 7. The increased chondrogenic response was associated with increased gene expression of Col2a1 and the protein expression of collagen II, a marker of cartilage formation at day 7. Sox9 has been used as a marker for chondrogenic differentiation 32, 33 and it was upregulated at day 7 in Osthole-treated group. However, treatment with Osthole did not affect the expression of Col2a1 and Sox9 in Bmp2Col2Cre KO mice at the same time point. Col10a1 is expressed in hypertrophic chondrocytes which could further undergo mineralization and apoptosis, connecting to subsequent angiogenesis and endochondral bone formation 34, 35. At day 10, the expression of Col10a1 was increased in Osthole-treated group, but not in Bmp2Col2Cre group.
We then analyzed expression levels of osteogenic marker genes, such as Runx2 and osteocalcin. Runx2 is a transcription factor playing important roles in chondrocytes and osteoblasts. Osteocalcin is mainly expressed in mature osteoblasts although it is also detected in hypertrophic chondrocytes. The expression of Runx2 and osteocalcin was increased at day 21 when mice were treated with Osthole. Interestingly, between the two groups with Osthole treatment, the relative level of Runx2 mRNA in Cre- control group were significantly up-regulated than that of Bmp2Col2Cre group at day 10. Besides we utilized biomechanical testing and assessed maximal torque, stiffness and toughness. Stiffness reflects the elasticity of bone while maximal torque and toughness represent the forces and energy required to disrupt the callus. In our study, the results of maximal torque, stiffness and toughness reveal that Osthole enhances the mechanical strength of the bone fracture healing.
The findings presented above are consistent with our hypothesis that Osthole plays an important role during fracture healing by accelerating endochondral bone formation, especially during the chondrocyte differentiation and maturation. BMP2 plays an important role in chondrocyte differentiation and maturation 21. BMP signaling pathway is known to regulate development and regeneration of bone and cartilage. BMPs elicit their function by binding to specific cell surface receptors with serine-threonine kinase activity and then inducing phosphorylation of receptor-regulated Smads (Smad1, 5, and 8) 36. Smad1/5/8 specifically transduces BMP signal, overexpression of Smad1/5/8 in chondrocytes promotes chondrocyte maturation. Maturation of chondrocytes is a marked step in ossification. BMP2 promotes Smad1/5/8 phosphorylation and mediates Runx2 expression 37. In our studies, the expression of pSmad1/5/8 was increased at day7 in Osthole-treated group, suggesting that Osthole promotes fracture healing through BMP2 signaling.
Therefore, we concluded that Osthole plays an important role in chondrocyte differentiation and maturation by regulating BMP2 signaling, and then influence endochondral ossification, thereby accelerating fracture healing.
This research has been partially supported by Zhejiang grants funded by Provincial Natural Science Foundation of China (Grant no. LY16H270010, LZ12H27001 and 81373669), the State Administration of Traditional Chinese Medicine of Zhejiang Province (Grant no. 2016ZA048), China Postdoctoral Science Foundation (Grant no. 2016M590533) and the Program for Zhejiang Leading Team of S&T Innovation and Key Laboratory of Zhejiang Province.