Effects of stepwise administration of osteoprotegerin and parathyroid hormone-related peptide DNA vectors on bone formation in ovariectomized rat model

Osteoporosis is a metabolic bone disease that impairs bone mineral density, microarchitecture, and strength. It requires continuous management, and further research into new treatment options is necessary. Osteoprotegerin (OPG) inhibits bone resorption and osteoclast activity. The objective of this study was to investigate the effects of stepwise administration of OPG-encoded minicircles (mcOPG) and a bone formation regulator, parathyroid hormone-related peptide (PTHrP)-encoded minicircles (mcPTHrP) in osteoporosis. The combined treatment with mcOPG and mcPTHrP significantly increased osteogenic marker expression in osteoblast differentiation compared with the single treatment groups. A model of postmenopausal osteoporosis was established in 12-week-old female rats through ovariectomy (OVX). After 8 weeks of OVX, mcOPG (80 µg/kg) was administered via intravenous injection. After 16 weeks of OVX, mcPTHrP (80 µg/kg) was injected once a week for 3 weeks. The bone microstructure in the femur was evaluated 24 weeks after OVX using micro-CT. In a proof-of-concept study, stepwise treatment with mcOPG and mcPTHrP on an OVX rat model significantly improved bone microstructure compared to treatment with mcOPG or mcPTHrP alone. These results suggest that stepwise treatment with mcOPG and mcPTHrP may be a potential treatment for osteoporosis.


Osteoblast differentiation of mcOPG-and mcPTHrP-transfected hiPSCs
To confirm the effects of mcOPG and mcPTHrP on in vitro osteogenesis, osteoblasts were differentiated from hiPSCs transfected with mcOPG, mcPTHrP, and mcOPG + mcPTHrP minicircles.The hiPSCs were transfected with the minicircles two days before osteoblast differentiation, and the cells were differentiated for 21 days (Fig. 2a).The relative expression of the pluripotency marker gene OCT4 was significantly decreased in the differentiated osteoblasts compared to its levels in hiPSCs (Fig. 2b).Furthermore, the expression of red and green fluorescent proteins (RFP) and GFP was verified in hiPSCs after transfection with the minicircles.The results were comparable to those observed in transfected HEK293T cells (Fig. 2c).The mRNA expression of PTHrP was higher in mcPTHrP and mcOPG + mcPTHrP-transfected hiPSCs than in non-transfected (control) and mcOPG transfected cells.(Fig. 2d) The mRNA expression level of OPG was higher in mcOPG and mcOPG + mcPTHrPtransfected hiPSCs compared with non-transfected and mcPTHrP-transfected cells (Fig. 2e).During in vitro culture, differentiated osteoblasts form mineralized nodules 34,35 , which were observed as brown and dark-colored nodules (indicated by white arrows) under the microscope on days 7, 14, and 21 of osteoblast differentiation (Fig. 2f).Mineralized nodule formation was most abundant on day 21, as confirmed by staining osteoblasts with alizarin red S (ARS), which specifically stains calcium deposits within mineralized nodules formed during osteogenic culture 36 .Mineralized nodules were observed in all groups (indicated by white arrows), with more nodules formed in the mcOPG + mcPTHrP group compared to other groups (Fig. 2g).Osteoblasts secrete and mineralize the extracellular matrix, which is primarily composed of collagen type I and contains smaller but significant amounts of other proteins, such as osteocalcin (OCN).OCN has been shown to promote mineral deposition and increases during matrix mineralization 37 .To assess osteoblast differentiation markers, we analyzed the relative gene expression of collagen type I (COL1A1) and OCN (Fig. 2h and i).The expression levels of osteocalcin (OCN) and collagen type I alpha 1 (COL1A1) were significantly higher in the group overexpressing both macrophage colony-stimulating factor (mcOPG) and parathyroid hormone-related protein (mcPTHrP)

In vivo delivery and expression confirmation of mcOPG and mcPTHrP
Previous studies have demonstrated that the generated minicircles promote in vitro osteogenesis.The highest differentiation efficacy was observed with the overexpression of both vectors.Before injecting the minicircles into disease model rats, we confirmed their efficacy in vivo using normal rats.To determine protein expression derived from mcOPG and mcPTHrP delivered to the kidney, spleen, and liver, two rats from each group were sacrificed on days 3, 7, and 15 after injection (Fig. 3a).Immunofluorescence staining confirmed OPG and PTHrP protein expression in rat tissues following minicircle injection (Fig. 3b-d, indicated by white arrows), as evidenced by GFP and RFP expression in each tissue.The successful in vivo delivery of mcPTHrP and mcOPG in the tissues of two individual rats at 3, 7, and 15 days after injection was confirmed by the increased gene expression of OPG and

Stepwise injection of mcOPG and mcPTHrP improves the bone microarchitecture in OVX
To confirm the effect of minicircles on in vivo bone regeneration, we generated osteoporosis rat models via OVX.After 8 weeks, mcOPG (80 μg/kg) was injected, followed by mcPTHrP (80 μg/kg) after an additional 8 weeks (Fig. 4a).We compared the stepwise injected group to each single injected group.We collected OVX rat femurs after 24 weeks for bone quality assessment, including BMD.A region of interest (ROI) was designated for assessing trabecular bone in the femur (Fig. 4b).Micro-CT images revealed that the microstructure of trabecular bone was improved with stepwise injections of mcOPG and mcPTHrP, compared to the other groups  www.nature.com/scientificreports/(see Fig. 4c).Furthermore, stepwise injection of mcOPG and mcPTHrP significantly increased BMD compared to the other groups (Fig. 4d).The stepwise injected group had a significantly higher bone volume fraction (BV/ TV) compared to the mcOPG and mcPTHrP injected groups (Fig. 4e).Trabecular number (Tb.N) only showed significance between the mcPTHrP injected group and the stepwise injected group (Fig. 4f).All groups showed similar levels in trabecular thickness (Tb.Th); however, the stepwise injected group showed a significant difference between the OVX control group and the mcPTHrP injected group (Fig. 4g).In summary, the mcOPG + mcPTHrP group showed significantly higher BMD, BV/TV, and Tb.N compared to the single injection groups (Fig. 4d-f).Furthermore, the mcOPG + mcPTHrP group had significantly higher Tb.Th compared to the mcPTHrP only group (Fig. 4g).Moreover, supplementary material containing additional parameters related to trabecular bone microstructure, such as bone surface (BS), total volume (TV), bone volume (BV), bone surface density (BS/TV), specific bone surface (BS/BV), trabecular separation (Tb.Sp), degree of anisotropy (DA), connectivity density (Conn.D), and structure model index (SMI), is provided (Supplementary Fig. 5).In addition, histological analysis of the bones was conducted and presented through hemoxylin and eosin (H&E) staining (Supplementary Fig. 5).
The results indicate that the stepwise injection of mcOPG and mcPTHrP enhances the quality and microstructure of trabecular bone in OVX rats.

Stepwise injection of mcOPG and mcPTHrP increase bone formation and inhibits bone resorption in OVX
The bone microstructure of OVX rats injected with both mcOPG and mcPTHrP was improved.To investigate the effects of the minicircles, we analyzed the expression of pro-osteogenic protein markers in the femurs of OVX rats injected with the minicircles.Compared to the OVX control group, the mcOPG, mcPTHrP, and mcOPG + mcPTHrP injected groups showed strong expression of RUNX2 (Fig. 5a).Collagen type I (COL1A1) expression levels exhibited a similar trend to that of RUNX2 in the mcOPG, mcPTHrP, and mcOPG + mcPTHrP injected groups (Fig. 5a and b).However, the protein expression of RUNX2 and collagen type I in the mcOPG + mcPTHrP group was not significant (Fig. 5c-e).
To confirm the effect of the minicircles on bone resorption, we conducted further experiments after inducing OPG expression in OVX rats, which is expected to block osteoclast differentiation.Cathepsin K (CatK) is mainly responsible for the degradation of collagen type I in osteoclast-mediated bone resorption and is predominantly www.nature.com/scientificreports/secreted by activated osteoclasts 38,39 .Interestingly, the expression of CatK and RANKL decreased in the mcOPG and mcOPG + mcPTHrP groups compared to the OVX and mcPTHrP groups.The mcOPG + mcPTHrP group had the lowest expression level of CatK (Fig. 6a) and a similar tendency was observed for RANKL (Fig. 6b).Furthermore, western blotting analysis revealed a slight decrease in bone resorption-related markers in the mcOPG and mcOPG + mcPTHrP groups (Fig. 6c-f).Moreover, the expression of OPG and RANKL proteins was lower in the mcOPG, mcPTHrP, and mcOPG + mcPTHrP groups compared to the OVX group (Fig. 6d and  e).The ratio of OPG to RANKL is crucial for osteoclast activity in the bone environment, with a higher ratio indicating lower differentiation and maturation of osteoclasts 33 .Although the OPG/RANKL ratio was higher in the mcOPG + mcPTHrP group than in the other groups, the difference was not statistically significant (Fig. 6f).
The results suggest that osteoclast activity is more effectively inhibited by stepwise injection of mcOPG and mcPTHrP compared to a single injection.Furthermore, OPG expression by mcOPG may have a stronger impact on osteoclast inhibition.

Discussion
Osteoporosis is a chronic disease with no cure that can completely reverse its impact.Currently, osteoporosis treatments include bisphosphonates, SERMs, denosumab, teriparatide, and romosozumab.Although these drugs are effective, their side effects limit their duration of use 40 .Therefore, research is needed to develop new drugs and strategies that use existing drugs and techniques more effectively.Various treatment options for osteoporosis and their effects are under study.Studies suggest that administering multiple drugs in combination may be more effective than using a single drug 12,41,42 .In this study, we examined the effect of stepwise treatment of OPG and PTHrP by injecting minicircle vectors, encoding OPG and PTHrP on bone regeneration in OVX rats.
Non-viral vector gene delivery is a promising and safe in vivo gene transfer strategy 43 .Plasmid DNA vectors can be effective; however, the remaining bacterial sequences may induce immune responses by generating antibodies against bacterial proteins.Minicircles are vectors that have had the bacterial backbone and transcription units, including antibiotic resistance genes, removed.Their relatively smaller size promotes the expression of foreign genes both in vitro and in vivo, making them potentially useful in preclinical gene therapy 44,45 .Minicircles were delivered in vivo via hydrodynamic tail vein injections to induce gene expression, which is a safe and efficient method for gene delivery 46 .Previous studies have confirmed the effectiveness of minicircle vectors www.nature.com/scientificreports/encoding human proteins [47][48][49][50] .In this study, we developed minicircle vectors encoding OPG and PTHrP (Fig. 1).
The transfected cells successfully induced self-production of OPG and PTHrP (Fig. 1e-g).
Several studies have investigated the effects of PTHrP on osteoblast differentiation, growth, and survival 51,52 .Furthermore, OPG participates in osteoclast differentiation, and its role in osteoblast differentiation remains controversial 53,54 .As osteoblast differentiation progresses, osteoblasts form more mineralized nodules 55 .The study confirmed an increase in mineralized nodule formation in osteoblasts differentiated from hiPSCs co-transfected with mcOPG and mcPTHrP (Fig. 2g).Moreover, the nodule formation of cells transfected with both minicircles was even greater than mcPTHrP alone.In addition, the cells co-transfected with both minicircles showed a high expression of COL1A1 and OCN, which are pro-osteogenic markers (Fig. 2h and i).The study confirmed the possibility that co-expression of OPG and PTHrP could improve osteogenesis, considering the degree of mineralization nodule formation and the expression of osteogenic markers.
This study suggested that OPG may have other biological roles in improving osteogenic differentiation and bone formation beyond its anti-osteoclastogenic role.OPG may have an anti-apoptotic role in reducing undesired cell death during bone generation within scaffolds, which could also protect newly synthesized mineral deposits 56 .In addition, OPG may recruit mesenchymal stem cells (MSCs) in synergy with bone morphogenetic protein-2 to enhance bone formation 57 .In a previous study conducted by Li and colleagues 56 , it was found that cell priming with OPG treatment during expansion culture increased the osteogenic differentiation ability of MSCs.Continuous treatment of OPG during both MSC expansion and osteogenic differentiation induced osteogenesis to a lesser extent compared to the OPG treatment used only in either cell expansion or differentiation culture.The authors discussed the potential for OPG to induce both anabolic and catabolic effects depending on the cell status.Furthermore, OPG is likely to prime undifferentiated human MSCs for enhanced osteogenesis.However, the mechanism by which co-expression of OPG and PTHrP affects osteoblast differentiation has not yet been revealed in this study.Based on the study by Li et al., NF-kB may be a possible candidate for increasing osteogenic differentiation.Confirming this intracellular molecule in future studies would be beneficial.Moreover, it would be interesting to confirm the effect of co-expressed OPG and PTHrP in osteoclasts or osteoblasts and osteoclast co-culture platforms to further confirm the synergetic effect of OPG and PTHrP in osteoblast and osteoclast activities and crosstalk.
The limitations of this proof-of-concept study must be considered when interpreting the results.The study used OVX modeling on rats to confirm the osteogenic effect of stepwise administration of mcOPG and mcPTHrP to an osteoporosis model.The OVX rat is a commonly used animal model for osteoporosis research due to its low cost and ease of handling.The study mainly focused on female rats as they are a specific model for postmenopausal human osteoporosis 58 .It has been reported that age-related bone loss in male SD rats begins mostly at 9 months of age, after bone growth has completed.Wang et al. suggest that male SD rats can be an appropriate animal model of age-related bone loss in men, as aging male rats showed comparable bone loss.To confirm the therapeutic effect of stepwise treatment of OPG and PTHrP, it is crucial to apply this strategy in male osteoporosis animal models.Furthermore, the small sample size and different number of animals per group limit the evaluation of the therapeutic effects of the suggested strategy.To evaluate the effect of stepwise administration of OPG-and PTHrP-encoding minicircles in vivo, a proof-of-concept study was conducted with a small sample size of 24 animals 6 per group.The minimum number of animals necessary for statistical analysis was used.To ensure humane treatment, rats were euthanized according to humane standards, such as complications due to aging, during the experiment.Some samples were damaged during the sample collection process and were eventually excluded from the project.Therefore, the final number of animals in each group used for analysis was as follows: (1) OVX group (n = 3), (2) OVX + mcOPG group (n = 4), (3) OVX + mcPTHrP group (n = 5), and (4) OVX + mcOPG + mcPTHrP (n = 6).The variation in the number of animals used in the analysis across groups may affect the validity of the statistical analysis.Thus, we intend to give this more consideration and study with a greater number of animals.
Determining the appropriate timepoint and dosage for administering mcPTHrP after mcOPG injection was a critical issue during this study.There are no confirmed reports on the effect of bone formation through stepwise administration in animal experiments, making it difficult to establish set standards.Because bone formation occurs after bone resorption during the bone remodeling process, it is assumed that mcOPG injection could suppress bone resorption and support bone formation.Additional bone formation is then promoted through mcPTHrP.Previous studies suggest that bone resorption occurs over 2 to 3 weeks, whereas bone formation occurs over 4 to 6 months during the bone remodeling process 59,60 .The administration period of mcOPG was determined based on the administration period of denosumab, a resorption inhibitor used to treat osteoporosis.Denosumab is typically administered once every six months in clinical settings 11 .However, we determined that a six-month gap was too long for rat animal testing and instead chose an eight-week gap.This was done to suppress bone resorption and provide a period of bone formation through mcOPG, regardless of age.Based on our previous studies, we selected a weekly injection period of three weeks for mcPTHrP injections, which have shown osteogenic effects 30 .We increased the dose of minicircles based on the body weight of the animals, as our previous research was conducted on mice.In future studies, we aim to confirm the doses and timepoints that can enhance the synergistic effects of mcOPG and mcPTHrP.Furthermore, a comparative analysis between the current group and a new group injected with mcOPG after mcPTHrP could provide further insight into the mechanism.
Although therapeutic options for osteoporosis have increased, no approved therapy has been found to fully restore normal bone integrity in patients 61 .Combination treatment with anabolic and antiresorptive agents has been suggested to improve treatment efficacy.However, attempts to combine teriparatide with bisphosphonates have failed to prove more consistent than monotherapy 62,63 .Recently, a meta-analysis study based on clinical trial publications [64][65][66][67][68][69] confirmed the increased efficacy of the combination of teriparatide and denosumab in treating both osteoporosis and postmenopausal osteoporosis.
In conclusion, this proof-of-concept study suggests a potentially new treatment strategy for osteoporosis: the stepwise in vivo delivery of OPG-and PTHrP-encoding minicircles that can induce self-production of protein.
Temporarily inducing the expression of OPG and PTHrP in vivo may be a potential gene therapy strategy for osteoporosis.This approach could prevent possible side effects caused by continuous expression while improving bone remodeling.The effect of stepwise injection of mcOPG and mcPTHrP on bone formation in an osteoporosis model was confirmed.The results showed that stepwise injection was more beneficial than a single injection of mcOPG or mcPTHrP.The sequential treatment may have promoted bone formation and inhibited bone resorption, ultimately improving bone quality and microstructure.

Minicircle production
Parental plasmids (OPG and PTHrP, mock RFP, and mock GFP) were purchased from System Biosciences (Palo Alto, CA, USA).The cDNA sequence of codon-optimized human OPG was subcloned into a mock RFP parental plasmid.The cDNA sequence of the codon-optimized human PTHrP (1-34 + 107-139) was subcloned into the mock GFP parent plasmid.These cDNAs were inserted into the XbaI and BamHI restriction sites of the multiple cloning sites downstream of the CMV promoter.Minicircle vectors were generated according to the manufacturer's instructions.Minicircle DNA vectors were extracted using NucleoBond Xtra plasmid purification kit (Macherey-Nagel, Duren, Nordrhein-Westfalen, Germany).The inserts encoded by the minicircles were confirmed by double digestion with XbaI and BamHI.

Minicircle transfection
Briefly, HEK293T cells were detached and seeded (3 × 10 5 ) in a 60 mm plate for transfection.The culture medium was replaced with serum and antibiotic-free DMEM one day prior to transfection.Cells were transfected with the minicircle vector using Lipofectamine 2000 (Thermo Fisher Scientific, Waltham, MA, USA), following the manufacturer's instructions.Successful transfection was confirmed the next day by detecting the expression of RFP and GFP in cells via fluorescence microscopy (Axio Observer.Z1 inverted, Carl Zeiss, Oberkochen, Germany).
hiPSCs were detached and seeded (3 × 10 4 ) in a 12-well plate pre-coated with 0.1% gelatin and serum media for transfection.hiPSCs were cultured in E8 and incubated at 37 °C in a 10% CO 2 atmosphere for 4 days, followed by transfection with the minicircle vector using the Lipofectamine 3000 reagent (Thermo Fisher Scientific, Waltham, MA, USA), according to the manufacturer's instructions.Successful transfection was confirmed the next day by detecting RFP and GFP expression in the cells using a fluorescence microscopy.

Osteoblast differentiation
Briefly, hiPSCs (3 × 10 4 ) were seeded in a 12-well plate pre-coated with 0.1% gelatin and serum media.hiPSCs were cultured in E8 and incubated 37 °C in a 10% CO 2 atmosphere for 4 days, followed by transfection.After 48 h, E8 medium was replaced with osteogenic differentiation medium (DMEM supplemented with 15% FBS, 100 nM of dexamethasone, 50 μg/mL of ascorbate-2-phosphate, 10 mM/L of β-glycerophosphate) to induce osteoblast differentiation 71 .The medium was changed every 2-3 days, and the cells were differentiated for 21 days, after which differentiated cells were harvested for quantitative reverse transcription polymerase chain reaction (qRT-PCR).

Alizarin red S staining
After being transfected with minicircle, osteoblasts were differentiated for 21 days.Following this, they were washed three times with PBS and fixed with 4% paraformaldehyde for 15 min at room temperature.The fixed cells were washed three times with deionized water (DW) and then incubated with 40 mM Alizarin Red S (ARS, ScienCell, Carlsbad, CA, USA) for 20 min at room temperature with gentle shaking.The stained cells were washed five times with DW and observed using an inverted routine microscope (Nikon Eclipse Ts2, Nikon, Tokyo, Japan).

Animal care and OVX model
This animal study complied with the ARRIVE 2.0 guidelines.All surgical interventions, including presurgical and postsurgical animal care, were performed under the Laboratory Animals Welfare Act, the Guide for the Care and Use of Laboratory Animals, and the Guidelines and Policies for Rodent Survival Surgery provided by the IACUC (Institutional Animal Care and Use Committee) at the School of Medicine, Catholic University of Korea (Approval number: CUMS-2021-0259-07).Female Sprague-Dawley (SD) rats, 8-week-old and body weights of 200-220 g, were purchased from Orient Bio, Inc. (Seongnam, Korea).After 1 week quarantine and adaptation period, the rats were bred for 3 weeks and used for experiments at 12-weeks of age.SD rats were housed at a temperature of 20-26 °C, humidity of 50 ± 10%, and a 12-h light-dark cycle.Food was provided as To confirm the effect of the minicircles on bone formation, OVX was performed on 18 (12-week-old) female SD rats to induce postmenopausal osteoporosis.SD rats were anesthetized by isoflurane via inhalation system and the target area was shaved, sterilized, and disinfected.To expose the ovaries, two dorsolateral incisions were made, and the ovaries were removed.The muscle and skin layers were sutured and disinfected.During anesthesia, Duratears Ophtalmic Ointment (Alcon Korea Ltd. 's, Seoul, Korea) was applied to the rats to prevent their corneas from drying out.In addition, their condition was monitored until they regained consciousness after surgery.After OVX, the rats were administered gentamicin (intramuscularly 5 mg/kg) and ketoprofen (intramuscularly 5 mg/kg) for 3-7 days.

Group allocation and minicircle injection
To confirm the expression of minicircles in normal SD rats, we intravenously injected mcOPG (80 μg/kg) or mcPTHrP (80 μg/kg) into 12-week-old female Sprague-Dawley rats (a total of 12 SD rats).The experimental groups were as follows: (1) the mcOPG group (n = 6), (2) mcPTHrP group (n = 6).We sacrificed two rats from each group on days 3, 7, and 15 after injection to analyze the protein expression of OPG and PTHrP in the kidney, spleen, and liver.
To confirm the pro-osteogenic effect of minicircles in OVX rats, the following procedures were performed on 24 SD rats randomly assigned 8 weeks after OVX induction and divided into six groups based on body weight (4 per group).One animal was randomly selected from each of the six cages, transferred to a new cage, and assigned a permanent number.The cages were then randomly assigned to each experimental group.This study investigates the effects of different treatments on a group of animals.The animals were divided into four experimental groups: (1) OVX group (n = 6), (2) OVX + mcOPG group (n = 6), (3) OVX + mcPTHrP group (n = 6), and (4) OVX + mcOPG + mcPTHrP (n = 6).The animals were randomly allocated to the groups by a third party.The veterinarians and breeders responsible for the care and treatment of the animals were not aware of the assigned group during the experiment.
After 8 weeks of ovariectomy (OVX), the rats were injected with mcOPG (80 µg/kg) through the tail vein.Similarly, after 16 weeks of OVX, mcPTHrP (80 µg/kg) was injected once a week for three weeks through the tail vein.The rats were sacrificed for histological and biochemical analysis after 24 weeks of OVX.The analysis excluded cases of euthanasia during the experiment and damage during sample collection after the animal experiment to humanely end the experiment.Therefore, the analysis was conducted on the following number of rats: (1) OVX group (n = 3), (2) OVX + mcOPG group (n = 4), (3) OVX + mcPTHrP group (n = 5), and (4) OVX + mcOPG + mcPTHrP group (n = 6).

Polymerase chain reaction (PCR)
Transfected cells were harvested and incubated with TRIzol reagent (Thermo Fisher Scientific, Waltham, MA, USA) to extract the mRNA.The kidneys, livers, and spleens of rats were collected at 3, 7, and 15 days after minicircle injection, homogenized using a homogenizer (Bio-Gen PRO 200 homogenizer, PRO Scientific, Oxford, Connecticut, USA) with TRIzol, and mRNA was extracted.cDNA was synthesized from extracted RNA using a RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, Waltham, MA, USA), and the target genes were amplified using a PCR system.Gene expression was normalized to that of GAPDH, and quantification was performed using image J software.The primers used for PCR are listed in Supplementary Table 1.

Quantitative reverse transcription polymerase chain reaction (qRT-PCR)
After 21 days of differentiation, the transfected hiPSC-derived osteoblasts were harvested, and cDNA was synthesized.qRT-PCR gene expression analyses were conducted on Light Cycler 480 (Roche Diagnostics, Indianapolis, USA) using LightCycler ® 480 SYBR Green 1 Master (Roche Diagnostics, Indianapolis, USA), according to the manufacturer's instructions.The expression levels of target genes were normalized to that GAPDH, and the relative expression was calculated using the ΔΔCt method.The OPG/RANKL ratio was determined using the Ct method.(The primers used for qRT-PCR are listed in Supplementary Table 2).

Western blotting analysis
Cells were harvested and lysed using radioimmunoassay precipitation buffer (RIPA, Sigma Aldrich, St. Louis, MO, USA) under constant shaking at 4 °C for 1 h, followed by centrifugation at 12,000 rpm for 20 min at 4 °C.For tissue samples, bones were harvested 24-weeks after OVX.Bones were homogenized and transferred into tissue protein extraction reagent (T-PER, Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 1 protease inhibitor cocktail tablet (Roche Diagnostics, Indianapolis, IN, USA) and 1 mM of PMSF under constant shaking at 4 °C for 2 h.The extracted protein samples were quantified using Bicinchoninic Acid (BCA, Thermo Fisher Scientific, Waltham, MA, USA) protein assay.Electrophoretic separation of proteins was performed using sodium dodecyl sulfate (SDS)-sulfate-polyacrylamide gels and transferred onto nitrocellulose blotting membranes (GE Healthcare, Chicago, Illinois, USA).Membranes were washed with Tris-buffered saline supplemented with Tween-20 (TBST) and blocked with 3% bovine serum albumin (BSA; Sigma Aldrich, St. Louis, MO, USA) or 3% non-fat milk (BD Difco, NJ, USA) with TBST.The membranes were incubated with specific primary antibodies (Supplementary Table 3) overnight at 4 °C.After washing, the membranes were incubated with the appropriate secondary antibodies.Protein expression was assessed using ECL solution (AbFrontier, Seoul, South Korea), followed by exposure of the membrane to the ImageQuant LAS 4000 system (BioRad, Hercules, CA, USA).Quantification of protein band intensity was performed using the ImageJ software.Band intensity of target proteins was normalized to that of GAPDH or β-Actin.

Figure 3 .
Figure 3. Expression of OPG and PTHrP-encoding minicircles in vivo.(a) Scheme of the injection of minicircles encoding OPG and PTHrP to rats via tail vein.(b-d) Immunofluorescence images of (b) spleen, (c) kidney, and (d) liver stained with OPG and PTHrP antibodies at 3, 7, and 15 days after intravenous (IV) injection with mcOPG and mcPTHrP via tail vein.Scale bar = 100 μm.(e-g) Relative expression of the OPG and PTHrP gene in (e) spleen, (f) kidney, and (g) liver.The two bands at each time point shown in the gel image represent the two rats analyzed at each time point.Results are presented as the mean band intensity normalized to that of GAPDH (Image J software).mcOPG minicircle encoding osteoprotegerin, mcPTHrP minicircle encoding parathyroid hormone-related protein, OPG osteoprotegerin, PTHrP parathyroid hormone-related protein.