Synergistic Bone Regeneration Through Sequential Dual-drug Delivery

Synergistic Bone Regeneration Through Sequential Dual-drug Delivery | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Synergistic Bone Regeneration Through Sequential Dual-drug Delivery Jae Won Jang, Hyunji Kim, Joon Seok Oh, Dongtak Lee, Hye-Jung Choi, and 12 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7438279/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract The global aging population has raised concerns regarding age-related health issues like osteoporosis and bone fractures. To address these conditions, bone-like scaffolds containing bioactive molecules and biomaterials have been widely studied. However, uncontrolled burst release and delivery of drugs can incur negative side effects. To overcome this issue, a collagen-hydroxyapatite scaffold (COHAS) that can sequentially deliver Bone morphogenetic protein-2 (BMP-2) and Osteoprotegerin fused to the Fc region of immunoglobulin (OPG-Fc) is synthesized. The COHAS comprises a collagen-hydroxyapatite matrix containing BMP-2 and numerous poly-lactic glycolic acid (PLGA) microspheres with OPG-Fc, dispersed in the matrix. The dispersion of PLGA microspheres enables the retardation of OPG-Fc release compared to BMP-2 release. The controlled sequential delivery of BMP-2 and OPG-Fc exhibits synergistic potential in promoting new bone formation by simultaneously activating osteoblasts and deactivating osteoclasts. This investigation revealed that the COHAS co-loaded with BMP-2 and OPG-Fc possesses excellent cell viability and enhanced osteogenic properties in vitro. In vivo assessment via implantation of the drug-loaded COHAS using an 8 mm-calvarial defect rat model demonstrated high efficacy of new bone formation with good biocompatibility. Hence, these findings provide valuable insights for developing therapeutic scaffolds capable of sequential release of multiple drugs, with the potential to extend a cell-free treatment system for bone regeneration. Biological sciences/Biotechnology Biological sciences/Drug discovery Physical sciences/Materials science Health sciences/Medical research bone regeneration sequential drug delivery bioinspired scaffold Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 1. INTRODUCTION As the global population continues to age, there is an increasing concern about age-related health issues such as osteoporosis and bone fractures 1 , 2 . Osteoporosis is a condition that weakens bones, making them fragile and prone to fractures. Fractures resulting from osteoporosis most often occur in the hip, spine, and wrist and can have serious consequences, including disability and even death 3 – 5 . Also, the large bone defects stemming from osteosarcoma, infections, and injuries from physical trauma should also be considered, which all demand a challenging process for treatment 6 . The conventional treatment methods, including autograft, allograft, and xenograft, have some drawbacks like limited obtainable quantities, immunogenic graft rejection, and nonunion of graft-host junction 7 . Furthermore, the cell-based bone repair method using stem cells requires prolonged time for healing and a high price 8 . To overcome these limitations, various strategies involving bioactive molecules and biomaterials are being explored to enhance bone regeneration by creating an optimal microenvironment that supports cell migration, proliferation, and differentiation 9 – 11 . For bone tissue regeneration, a three-dimensional scaffold with a porous architecture plays a crucial role by providing a supportive environment that accommodates preosteoblasts or bone marrow mesenchymal stem cells and promotes their differentiation into mature cells. 12 – 16 . Collagen and hydroxyapatite, the main components of natural bone, are widely used biomaterials for bone regeneration scaffolds 17 – 19 . Their availability and versatility enable the the mass production of bone scaffolds, offering a promising alternative to existing bone graft materials 20 . Collagen shows excellent cell adhesion properties by providing numerous binding sites for cell membrane receptors, which contribute to high osteoinductivity 21 . However, substrates composed solely of collagen have poor mechanical strength, rendering them insufficient to maintain structural integrity at bone defect sites 22 . Hydroxyapatite, a well-known osteoconductive biomaterial, can be incorporated to enhance structural support by increasing mechanical strength and to promote bone regeneration by releasing bone-forming ions and bioactive molecules in a controlled manner 23 . Bone morphogenetic protein-2 (BMP-2), an osteoinductive growth factor approved by the Food and Drug Administration (FDA), is widely recognized as a key biological agent encapsulated within scaffolds for bone regeneration 24 . BMP-2 is produced by osteoblasts and plays a critical role in multiple stages of bone formation, including osteoblast proliferation and differentiation, bone matrix production and mineralization, and bone tissue remodeling 25 . However, its high dose and uncontrolled release can lead to potential complications, including heterotopic ossification, tissue inflammation, and the induction of osteoclastogenesis multiple molecular pathways 24 , 26 , 27 . Therefore, careful optimization of dosing and delivery methods is essential to maximize the therapeutic benefits of BMP-2 while minimizing its associated risks. Osteoprotegerin (OPG), another FDA-approved biological agent used in bone regeneration, is an innate protein that regulates bone metabolism by inhibiting the receptor activator of nuclear factor-κB ligand (RANKL) 28 . In particular, the Fc region is added to OPG to create OPG-Fc, which extends the protein’s half-life 29 . OPG-Fc promotes new bone formation by suppressing osteoclast differentiation and activity. Both biological agents have demonstrated effectiveness in promoting bone formation in preclinical and clinical studies involving various bone disorders, including osteoporosis, bone metastasis, and fractures 30 , 31 . Previous studies have explored the combined use of BMP-2 and OPG to enhance the bone healing process. For instance, Bougioukli et al. demonstrated advancements in new bone formation and integration at the site of a mouse critical-sized femoral defect by delivering BMP-2 and OPG-Fc 32 . OPG-Fc was systemically administered through subcutaneous injection. A notable limitation of this approach is the absence of a drug carrier for OPG-Fc delivery, which could potentially lead to osteopetrosis due to elevated drug absorption. Wang et al. showed improved osseointegration in an osteoporotic animal model by employing 3D-printed composite scaffolds loaded with BMP-2 and OPG 33 . Wei et al. utilized a collagen sponge patch loaded with BMP-2 and OPG in a rabbit joint surgery model, leading to enhanced tendon-bone healing 34 . Both studies confirmed the sustained release of BMP-2 and OPG; however, they were limited to simultaneous release only and lacked a mechanism for sequential, delayed release that reflects the entire bone regeneration process. In the natural bone healing process, the initial stage involves the differentiation of the osteoclast precursors following the differentiation and activation of osteoblasts 35 . The osteoclast cycle begins approximately five days after the onset of the bone defect. During this cycle, osteoclasts resorb bone by secreting hydrogen ions and proteolytic enzymes into the local microenvironment. This process typically lasts for about two to three weeks. The activation of osteoclasts during the early stage helps remove debris and residual material from the site of bone damage, creating an optimal environment for bone formation. However, if osteoclasts remain persistently active beyond this stage, they may impede bone formation. Therefore, inhibiting osteoclastogenesis after the early stage is necessary to enhance the effectiveness of bone regeneration. In this context, the simultaneous release of a bone growth factor and osteoclastogenesis inhibitor may reduce the efficacy of new bone formation. The inhibitor may unnecessarily suppress osteoclast activity during the early stage, while its short half-life limits its effectiveness during the intermediate stage. Accordingly, we propose that sequential delivery—specifically, delayed release of the osteoclastogenesis inhibitor following the bone growth factor—represents a promising strategy for enhancing bone regeneration. The delayed release of osteoclastogenesis inhibitor is thought to prevent unnecessary inhibition during the early stage. In this regard, Lee et al. demonstrated that sequential release of the BMP-2 and alendronate (i.e., delayed release of alendronate) can significantly enhance new bone formation 36 . Alendronate is a small-molecule osteoclast inhibitor and a common therapeutic agent for osteoporosis. Building on these findings, we speculate that delayed release of biological agents such as antibodies (e.g., OPG-Fc) can also effectively inhibit osteoclast activity after the early stage, leading to more efficient bone regeneration. Investigating the clinical impact of delayed release of biological inhibitors on bone regeneration is therefore important. However, studies dealing with this strategy—particularly, the delayed release of antibodies—have yet to be published. In our study, we engineered a cell-free bone regeneration scaffold, the collagen-hydroxyapatite scaffold (COHAS), incorporating BMP-2 and OPG-Fc to enhance bone regeneration (Fig. 1 ). This scaffold mimics natural bone and serves as an alternative to bone grafts, offering excellent osteoconductivity. It also acts as a reservoir for two factors: BMP-2 and OPG-Fc (Fig. 1 a). We hypothesized that sequential release of BMP-2 and OPG-Fc would have a synergistic effect on bone regeneration. The initial release of BMP-2 triggers osteogenic differentiation of mesenchymal stem cells (MSCs), promoting osteogenesis. Subsequently, OPG-Fc is released to inhibit osteoclast differentiation by binding to RANKL, thereby further enhancing bone regeneration (Fig. 1 b). We assessed the in vitro and in vivo effects of this sequential release and compared them with single or simultaneous release of BMP-2 and OPG-Fc, as summarized in Table 1 . Our results demonstrated that sequential release significantly improved bone regeneration with minimal inflammation at 8 week post-implantation in an 8 mm calvarial defect rat model (Fig. 1 c). 2. MATERIALS AND METHODS 2.1 Materials Dulbecco’s modified eagle’s medium (DMEM) (Corning, USA), fetal bovine serum (Welgene, South Korea) and penicillin-streptomycin (Corning, USA) were used to culture preosteoclast cell. Alpha-MEM (α-MEM) (Welgene, South Korea), fetal bovine serum and penicillin-streptomycin were used to culture preosteoblast cell. We purchased soluble Receptor Activator of NFkB Ligand (sRANKL) from PROSPEC Protein Speciallists (USA), recombinant human bone morphogenetic protein-2 (rhBMP-2) from Peprotech (USA), carboxyl-terminated 50:50 PLGA (0.67 dl/g) from LACTEL Absorbable Polymers (USA), collagen Type 1 Rat Tail of 3.8 mg/mL from Corning (USA) and hydroxyapatite nanopowder(< 200nm particle size) from Sigma Aldrich (USA). Human Osteoprotegrin/TNFRSF11B Duoset ELISA and human BMP-2 ELISA kit were purchased from R&D systems (USA) and ABclonal (USA). The cell counting-8 kit (CCK-8) was purchased from Merck KGaA (Germany). Tartrate-resistant acid phosphatase assay (TRACP) kit and alkaline phosphatase assay (ALP) kit was purchased from Takara Bio (Japan). Preosteoblast MC3T3-E1 cells, human osteoprotegerin (OPG-Fc) were provided by Eulji Medical Center. 2.2 Fabrication of collagen-hydroxyapatite scaffold A collagen-hydroxyapatite scaffold was fabricated following a previous method with minor modification 36 . Briefly, COHAS slurry was prepared by incorporating 100 mg hydroxyapatite powder into the 2 ml collagen matrix. All procedures were carried out at a temperature of 4°C. To ensure a homogeneous mixture, the slurry was subjected to high-speed homogenization at a minimum of 10,000 rpm for a duration of 5 minutes. Subsequently, a degassing process was employed to eliminate any undesired bubbles. The degassed slurry was then combined with a neutralization solution within an 8 mm diameter polydimethylsiloxane (PDMS) mold and stirred. The resulting mixture was incubated at 37°C for 1 hour and subsequently transferred to a deep-freezer set at -80°C. The frozen scaffold was subjected to freeze-drying to obtain the final COHAS. Prior to use, the scaffold was sterilized using UV light (λ = 254 nm) for a duration of 4 hours. 2.3 Mechanical property characterization of collagen-hydroxyapatite scaffold Compressive strength testing was conducted using a universal testing machine (UTM; ST-1000, Salt Co., Ltd., Incheon, Republic of Korea). Cylindrical scaffold specimens, prepared in the swollen state after immersion in phosphate-buffered saline (PBS), had an average diameter of approximately 8 mm and a height of approximately 5 mm. Three distinct specimens were tested for statistical reliability. All tests were performed under displacement control at a crosshead speed of 1 mm/min. A preload of 0.1 N was applied before measurement, and the compressive stress–strain curves were obtained from the recorded load–displacement data. Stress was calculated by normalizing the compressive load to the initial cross-sectional area, and strain was determined from the change in specimen height relative to the initial height. The compressive strength was defined as the average stress within the plateau region, corresponding to a strain range of 10–30%. The porosity of the freeze-dried collagen-hydroxyapatite scaffolds was determined from the measured scaffold density and the theoretical composite density. The mass of each dried scaffold (n = 8) was measured using a digital balance (PioneerTM, OHAUS Co., USA; accuracy: ± 0.0001 g), and their volume was determined from its dimensions obtained using a digital Vernier caliper (CD-15APX, Mitutoyo Co., Japan; accuracy: ± 0.01 mm). The apparent scaffold density (ρ_scaffold) was calculated as: $$\:{\rho\:}_{scaffold}=\frac{{m}_{scaffold}}{{V}_{scaffold}}$$ where \(\:{m}_{scaffold}\) is the dried scaffold mass and \(\:{V}_{scaffold}\) ​ is the volume of scaffold. The theoretical density of the solid composite \(\:\left({\rho\:}_{material}\right)\) was calculated from the masses of collagen and hydroxyapatite used and their theoretical densities, assumed to be 1.23 g/cm³ and 3.14 g/cm³, respectively. The percent porosity (%) was then determined as, $$\:Porosity\:\left(\%\right)=\left(1-\:\frac{{\rho\:}_{scaffold}}{{\rho\:}_{material}}\:\right)\:\times\:100$$ 2.4 Synthesis of PLGA microsphere containing drugs PLGA microsphere was synthesized using oil-in-water (O/W) technique 36 . To prepare the PLGA-based formulation, 100 mg of PLGA polymer was dissolved in 2 ml of dichloromethane through stirring. rhBMP-2 and OPG-Fc were separately added to the PLGA solution. The resulting PLGA solution was then introduced dropwise into a stirring 0.5% polyvinyl alcohol (PVA) solution. The mixed solution was homogenized at 3,000 rpm for 30 seconds. The emulsion obtained from the homogenization step was stirred using magnetic stirrer to facilitate vaporization of organic solvent. Subsequently, the mixture was transferred to a conical tube and centrifuged at 3,000 rpm for 3 minutes. The supernatant was carefully collected and washed 3 times with distilled water. 2.5 Drug release kinetics of OPG-Fc The drug release kinetics of OPG-Fc was evaluated by a reported method 36 . In brief, the COHAS samples were placed in separate wells of a 6-well plate. The total amount of OPG-Fc was 10 µg in each sample. Each well contained 2 ml of PBS. The plate was then subjected to shaking at 37°C for the duration of the incubation period (WIS-20, Daihan Scientific, Korea). The solution was extracted from the 6-well plate at given time points and re-filled with same amount of PBS. The release profile was examined by OPG-Fc ELISA (DuoSet ELISA, R&D systems, USA) according to the protocol provided from manufacturer via a microplate reader (SpectraMax ABS Plus, Molecular Devices, CA, USA). The standard graph was determined using the microplate reader set to 450 nm. Release kinetic of OPG-Fc was quantified by the same procedure. In addition, we defined the apparent encapsulation efficiency (EE_app) as the cumulative amount released at the plateau (day 56) divided by the initial input (10 µg) × 100. We note that this EE_app represents a conservative lower-bound estimate of the true encapsulation efficiency because incomplete release and potential analytical loss may lead to underestimation of the encapsulated amount. 2.6 Drug release kinetics of BMP-2 The drug release kinetics of BMP-2 was evaluated by a reported method 36 . The hydrogels were placed individually in 6-well plate with 2 ml of PBS. 1 µg of BMP-2 was contained in each hydrogel and the hydrogels were incubated in 37°C shaking incubator (WIS-20, Daihan Scientific, Korea). We extracted the solution at fixed times and replaced it with fresh PBS. The drug concentration was examined by human BMP-2 ELISA kit (ABclonal, MA, USA) by provided protocol. The release kinetics and the standard graph were measured using the microplate reader set to 450 nm and the wavelengths were corrected with 630 nm for optical imperfections. 2.7 In vitro Tests The preosteoclast RAW 264.7 cells were cultured in DMEM supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin. The cell culture was maintained in a 5% CO 2 atmosphere at 37°C. TRACP assay kit was performed to confirm the osteoclast differentiation inhibitory effect of OPG-Fc. 5 x 10 3 cells were incubated in each well of 96-well plate and 50 µg/ml of sRANKL was contained to promote osteoclast differentiation. The 200 µl of media was added for cell culture and changed with appropriate medium every 2 days. The cell differentiation was examined at 1, 3, 7 days and the solution was diluted 20-folded for measurement. The absorbance of groups was measured with microplate reader. The preosteoblast MC3T3-E1 cells were cultured in alpha-minimum essential medium (α-MEM) supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin in a 5% CO 2 at 37°C. To assess cell proliferation, a Cell Counting Kit-8 (CCK-8) assay was performed following the manufacturer's protocol. Briefly, the MC3T3-E1 cells were seeded at a density of 5 x 10 3 cells per well in a 96-well plate containing COHASs incorporated with 1 µg of rhBMP-2 and 10 µg of OPG-Fc. The cell viability was examined using a microplate reader at days 1, 3, and 7. The cell culture medium in each well was changed every two days. The differentiation of cells was evaluated via ALP assay at 1, 3, and 7 days. The intensity of the resultant color was measured in a dark setting to accurately quantify the ALP activity. 2.8 In vivo study All animal experiments were approved by the Institutional Animal Care and Use Committee in Seoul National University Hospital (SNUH-IACUC) with IACUC No. 21-0202-S1A) and animals were maintained in the facility accredited AAALAC International (#001169) in accordance with Guide for the Care and Use of Laboratory Animals 8th edition, NRC (2010). Eighteen healthy male Sprague-Dawley rats (2 months old, weighing 250–300 g) supplied by Koatech, Keung-Ki, Pyong-Taek, Korea, were used for the study. Each rat was assigned a unique ID and randomly allocated into one of six experimental groups (n = 3 per group). The scaffold was fabricated with 1 µg of BMP-2 and 50 µg of OPG-Fc for in vivo experiment. We selected animals with similar age, sex, and body weight for the experiments and assigned each animal a unique ID before randomly allocating them into experimental groups. The surgeon was aware of the group assignments during the procedure; however, the outcome evaluator was blinded to group allocation and assessed the results based solely on the animal IDs. This blinding approach minimized potential bias and ensured the objectivity of the outcome assessment. For critical size calvarial defect model, the animals were anesthetized by using 2–5% isoflurane for induction and 0.25 ~ 0.4% for maintenance. Their scalp was shaved from the end of the skull to the midpoint between the eyes using electric clippers. Betadine (Hyundai Pharm, Korea) were used for disinfection. With a no.15 blade, an incision sized 4 cm was made down to bone level. Periosteum was dissected laterally to expose the underlying skull using an elevator. Trephination using 8 mm-sized electrical burr (Seoul, South Korea) was done to make the bone defect. Periosteum was closed with running 4 − 0 Monocryl suture. Skin closure was done using running suture with 4 − 0 Prolene. Bone regeneration was evaluated using in vivo micro computed topography (µ-CT; NFR Polaris-G90, Nano Focus Ray, Korea) with isotropic voxel size of 9 µm and X-ray tube voltage at 55kV and current at 180 µA. Imaging was done under anesthesia using isoflurane at 2, 4, and 8 weeks after COHAS insertion. At each point the regenerated bone volume and surface area were evaluated and calculated using Analyze (Version 12.0, AnalyzeDirect, Inc. USA). For histopathological examinations, hematoxylins-eosin (H&E) and Masson’s trichrome (MT) staining methods were conducted. The skull tissue encompassing both the defect and the surrounding normal bone was collected after an 8-week period. Subsequently, the tissue samples were fixed in 10% buffered formalin for 3 days, decalcified for 7 days, and sectioned into 5 µm thick slices. The samples were examined under a light microscope (BX53F, OLYMPUS, Japan) photographed at ×40, ×100, and ×400 magnifications. This study was reported in accordance with the ARRIVE guidelines. 2.9 Statistical Analysis All statistical analysis was performed by the Medical Research Collaborating Center at Seoul National University Hospital using SAS statistical software (SAS system for Windows, version 9.3; SAS Institute, Cary, NC, USA). A mixed model analysis was performed to test for differences in bone formation area according to drug combinations, accounting for repeated time measurements. The interaction between drug combination and time point was found to be non-significant and was therefore removed from the model. At the 8-week time point, a mixed model analysis including the group-time interaction term was conducted to examine differences between COHAS and COHAS_B_P@O, and between COHAS_B_P and COHAS_B_P@O, for bone regeneration area and volume. A linear mixed model was used to account for variance from repeated measurements over time. Multiple group comparisons were performed without correction for increased Type I error, due to the exploratory nature of this animal study. Although up to 15 pairwise comparisons were possible among the six groups (6C2 = 15), the main comparisons of interest were predefined. For transparency, exact p-values and 95% confidence intervals are reported in Supplementary Tables S2–S6. Significant statistical differences were indicated as *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001. 3. RESULTS 3.1. Morphological Analysis and Mechanical Property Characterization of Scaffold The morphology and dimensions of the hydroxyapatite-based scaffold were examined using scanning electron microscopy (SEM). To achieve sequential delivery of BMP-2 and OPG-Fc, we fabricated PLGA microspheres and a drug-loading scaffold based on our previous study. The scaffold, COHAS, consisted of collagen and hydroxyapatite. Their ratio was carefully optimized to support effective drug delivery. Table 1 lists the abbreviations for scaffold samples with different combinations of BMP-2, OPG-Fc, and PLGA microspheres. The cross-section of the lyophilized collagen hydrogel showed a porous structure with a large surface area, suitable for accommodating hydroxyapatite (Figure S1 , Supporting Information). Compared to scaffolds made solely of hydroxyapatite or collagen, COHAS displayed a distinct, more intricate structure, as shown in Fig. 2 a. The average size of the PLGA microspheres containing OPG-Fc (P@O) was approximately 100 µm, indicating a microscopic scale (Fig. 2 b). These microspheres possess a porous structure that allows drug loading and can be modified to control drug release 37 . Figure 2 c shows the inner section of COHAS_B_P@O, where the collagen matrix and PLGA microspheres coexist. Since OPG-Fc is encapsulated within the PLGA microspheres dispersed in COHAS, it must pass through two barriers before reaching the external environment. Thus, in COHAS_B_P@O, OPG-Fc release follows the release of BMP-2, ensuring sequential delivery. The compressive strength of the COHAS was 2.80 ± 0.17 kPa calculated from Figure S2. This value is within the typical range for COHASs used in bone tissue engineering. Many works note that optimal osteoblast proliferation and maturation can happen on matrices softer than bone itself, often below 10 kPa 38 , 39 . Some evidence indicates that softer scaffolds (~ 1–3 kPa) can even enhance osteogenic differentiation at early stages compared to harder substrates 40 . Therefore, these previous reports support that the compressive strength of our scaffold is suitable for bone repair, particularly during the initial phases of bone healing. The percent porosity of the scaffold was 95.82% which measured from eight COHASs, using the equation (Table S1 ). Porosity above 90% is often targeted in bone scaffolds because it provides a large surface area and interconnected pore network that facilitates cell adhesion, migration, nutrient and oxygen diffusion, and vascularization. These features support robust bone tissue regeneration 41 – 43 . Therefore, the high porosity of our scaffold is considered advantageous for bone tissue regeneration. Table 1 Abbreviations for the various samples used in the study. Name of sample Description of sample COHAS collagen-hydroxyapatite scaffold P@O PLGA microspheres containing OPG-Fc COHAS_B scaffold incorporated with BMP-2 COHAS_O scaffold incorporated with OPG-Fc COHAS_B_O scaffold incorporated with BMP-2 and OPG-Fc COHAS_P@B_O scaffold incorporated with BMP-2 encapsulated in PLGA microspheres and OPG-Fc COHAS_B_P@O scaffold incorporated with BMP-2 and OPG-Fc encapsulated in PLGA microspheres 3.2. In Vitro Release of OPG-Fc and BMP-2 from COHAS Accurately monitoring the concentration of bioactive molecules in vivo is nearly impossible. Therefore, we investigated the release patterns of BMP-2 and OPG-Fc from the scaffolds in vitro by simulating body fluid conditions. The Scaffolds were immersed in phosphate-buffered saline (PBS), which has ionic concentration and osmotic pressure similar to human body fluid and helps maintain stable pH. We quantified OPG-Fc release from two COHAS groups, COHAS_O and COHAS_P@O, using OPG-Fc ELISA kits (Fig. 3 a). COHAS_O showed burst release behavior, with a significant amount of OPG-Fc released by day 8. In contrast, COHAS_P@O exhibited a delayed release profile. It steadily released the drug until day 42 and reached saturation by day 56 (Fig. 3 c). This delayed release is due to the encapsulation of OPG-Fc within PLGA microspheres. These microspheres slow the diffusion of the drug from COHAS to the external environment. The cumulative amounts of OPG-Fc released from COHAS_O and COHAS_P@O differed by only 7.13% at day 56. This indicates that OPG-Fc was effectively encapsulated in the PLGA microspheres and demonstrated the stable release performance. The loading efficiency of OPG-Fc was approximately 88.2% for COHAS_O and 81.9% for COHAS_P@O, based on the released amount relative to the initial loading. These results indicate a notably higher drug loading rate compared to previous studies 44 . They also suggest that OPG-Fc has minimal interaction with either COHAS or PLGA. The inhibitory effect of OPG-Fc on preosteoclast differentiation, specifically osteoclastogenesis, was tested by adding varying concentrations of OPG-Fc to RAW264.7 cells in the presence of the osteoclast differentiation factor, soluble RANKL (sRANKL), as depicted in Fig. 3 b. Osteoclastic differentiation was measured using the tartrate-resistant acid phosphatase assay (TRACP) assay kit. Compared to the sRANKL-only group, all OPG-Fc treated groups (10, 50, 100 µg) showed consistent inhibition on both day 3 and day 7. Notably, on day 7, the inhibition rates increased with OPG-Fc concentration, reaching 84.0%, 93.0%, and 97.2% for 10 µg, 50 µg, and 100 µg, respectively. The BMP-2 release profile of COHAS_B was measured using BMP-2 ELISA kits (Fig. 3 d). The release patterns of BMP-2 from both COHAS_B and the COHAS_P@B were similar to those shown in Fig. 3 c. Specifically, COHAS_B exhibited a burst release, with about 50% of the total BMP-2 released on day 1 and approximately 72% by day 14. In contrast, COHAS_P@B showed a delayed release, reaching around 73% release only by day 42. This delay is attributed to BMP-2 encapsulation within PLGA microspheres. 3.3. In Vitro Cell Proliferation & Osteogenesis study To assess the cytotoxic effects of various COHAS groups, a cell viability assay was performed using the cell counting kit-8 (CCK-8) on days 1 and 7. The control consisted of cells incubated without COHAS. On day 7, all COHAS groups were confirmed to be non-toxic and did not affect cell growth (Fig. 4 a). This finding also suggests that both bioactive agents, BMP-2 and OPG-Fc, have excellent biocompatibility (Figure S3a, Supporting Information). The osteogenic effects of the COHAS groups were evaluated via alkaline phosphatase (ALP) assay kits. The ALP activity of COHAS_O on day 7 exhibited minimal change compared to day 1, indicating that OPG-Fc alone is insufficient to activate preosteoblasts (Fig. 4 b). This is supported by data in Figure S3b, which demonstrates that preosteoblasts did not differentiate or activate when directly exposed to OPG-Fc. COHAS_B_O showed a greater increase in ALP activity compared to COHAS_B, suggesting a potential synergistic effect from the simultaneous application of BMP-2 and OPG-Fc. Moreover, COHAS_B_P@O demonstrated the highest ALP activity among all groups. This is likely due to the delayed release of OPG-Fc and its synergistic interaction with BMP-2 (Fig. 4 b). 3.4. In Vivo Study To assess bone regeneration, a critical-size calvarial defect rat model was used. A preformed COHAS was carefully inserted into the defect area, as shown in Figure S4. The animals were divided into six groups, each consisting of three rats. The calvarial defects were filled with different materials: COHAS, COHAS_B, COHAS_O, COHAS_B_O, COHAS_B@P_O, and COHAS_B_P@O. Figure 5 a presents 3D reconstructed image illustrating bone regeneration in all groups. Bone growth was observed in both the central and peripheral regions of the defect site. The average area and volume of regenerated bone for each group are summarized in Fig. 5 b, Fig. 5 c, and Table S2. The results revealed clear variations in bone regeneration among the groups. COHAS had the smallest new bone area and volume, while COHAS_B_P@O demonstrated the highest level of regeneration. Tables S3 and S4 provide a comprehensive comparison of area and volume measurements, using COHAS as the reference. Importantly, COHAS_P@B_O exhibited a modest enhancement in bone growth compared to COHAS_B_P@O. This highlights the significant impact of drug release sequence on bone regeneration efficiency. Analysis of absolute area and volume measurements revealed statistically significant differences among the various groups (p = 0.0262 for area and p = 0.0170 for volume). Specifically, COHAS_B_P@O displayed a significantly larger increase in bone regeneration area (p = 0.0076) and volume (p = 0.0019). In contrast, COHAS_B_O had smaller increases in both area and volume. Considering the delayed release kinetics of OPG-Fc, which saturates by week 8 (Fig. 3 c), these result support the hypothesis that sequential release of BMP-2 and OPG-Fc from COHAS enhances bone healing at the defect site. Other groups, except for the volume in COHAS_P@B_O (p = 0.0483), did not show statistical significance (p > 0.05). Temporal analysis further demonstrated that COHAS_B_P@O had a significant increase in bone area and volume at both weeks 4 and 8 compared to week 2 (all p-values < 0.05). Histological examination of the calvarial defect site using H&E and MT stains is presented in Fig. 6 a. The MT staining method highlights non-mineralized osteoid connective tissue in blue, while mineralized bone appears red. Among the experimental groups (COHAS, COHAS_B, COHAS_O, COHAS_B_O, COHAS_P@B_O), pronounced infiltration of inflammatory cells was observed in the central defect region. In contrast, the COHAS_B_P@O group showed minimal inflammatory cell presence. Although the extent of bone maturation varied across groups, all exhibited some degree of maturation resembling normal bone structure. Notably, the COHAS_B_P@O group displayed the highest level of bone maturation, as indicated by the larger red-stained area in the MT staining (Fig. 6 b). 4. Discussion To date, an effective approach to treat critical bone defects remains elusive. Autologous bone transplantation is considered the gold standard for bone regeneration. However, its widespread application is limited by significant time and cost constraints 45 . Moreover, fractures in the elderly are expected to increase due to the global aging population. This trend is accompanied by a rise in age-related bone diseases such as osteopenia, osteoarthritis, and osteoporosis 46 . Hence, there is a pressing need for alternative, more practical solutions for bone healing. During post-fracture bone healing, the inflammatory response remains active for up to five days. This phase is followed by osteoblast activity, which lasts for about ten days. Afterward, osteoclasts collaborate with osteoblasts to remove residual material. Maintaining a balanced ratio between osteoclasts to osteoblasts is crucial for bone health. In patients with osteoporosis, however, the proportion of osteoclasts is elevated relative to osteoblasts. Because of this imbalance, inhibiting osteoclasts is a well-established strategy to prevent osteoporosis. Many studies suggest that bone-replaceable scaffolds, designed to mimic the natural bone healing process, can enhance new bone formation and mitigate associated side effects 47 . In these studies, scaffolds loaded with bioactive molecules have been developed to support bone healing by releasing essential components. However, simultaneous release of these factors may not provide sufficient bone healing efficacy and could even be harmful. Bone regeneration requires about two weeks for optimal osteoblast activation. Therefore, BMP-2 needs both an initial burst release and sustained delivery. Previous research has mainly focused on temporal release, but bulk drug administration has sometimes caused ectopic bone formation 48 – 50 . Additionally, administering OPG-Fc alone has shown limited success in promoting bone generation 32 . To address these issues, we developed scaffolds that sequentially release BMP-2 and OPG-Fc. This approach aims to synergistically improve bone regeneration. The key component enabling the delayed release of bioactive molecules is PLGA. It is widely used as a drug carrier due to its biodegradable and biocompatible properties 51 . We confirmed that BMP-2 and OPG-Fc were successfully incorporated into PLGA microspheres, with encapsulation rates of 86.9% and 81.9%, respectively. In Figs. 3 c and 3 d, we compared the total released amounts of BMP-2 in COHAS_B and COHAS_P@B, as well as OPG-Fc in COHAS_O and COHAS_P@O. This analysis verified that the COHAS components, collagen and hydroxyapatite, do not interfere with PLGA or impede the release of BMP-2 and OPG-Fc. Among the experimental groups shown in Fig. 5 , COHAS_B_P@O achieved the most notable result, demonstrating exceptional new bone formation through the delayed release of OPG-Fc. The release profile of OPG-Fc was more delayed compared to that of BMP-2. This difference arises because OPG-Fc is encapsulated within both PLGA microspheres and COHAS, whereas BMP-2 is embedded only in COHAS. Especially, the absolute area and volume of new bone in COHAS_B_P@O exceeded those in COHAS_P@B_O. This result highlights the importance of the drug release sequence in the bone healing process. It also aligns with previous studies regarding the biological stages of fracture healing 35 . Specifically, the initial release of BMP-2 stimulates the preosteoblasts. The delayed release of OPG-Fc then inhibits osteoclastogenesis of presoteoclasts in the bone defect microenvironment. Figure S3 shows that only BMP-2 increases ALP activity in MC3T3-E1 cells, while OPG-Fc does not. This is consistent with the results presented in Fig. 5 . Furthermore, COHAS_B exhibited significantly greater absolute bone area and volume compared to COHAS_P@B_O. This highlights the importance of properly configuring the release sequence. An improper sequence of BMP-2 and OPG-Fc may negatively affect bone regeneration, potentially more than releasing BMP-2 alone. Our study focuses on the delivery of multiple drugs and holds significant potential for broader applications in the therapeutic applications, such as wound healing. However, there are limitations to the use of COHAS. Firstly, the bone defect model we used targets cranial bone defects. These defects have minimal load-bearing requirements and lack motion compared to joints or other mobile body parts. In future studies, we plan to enhance the applicability of COHAS by combining it with supportive structures, such as metal frameworks, to provide mechanical strength for load-bearing sites like the knee and spine. Second, the optimal drug loading amount should be customized to suit different bone defect cases, considering variations in size, depth, and severity. Accordingly, we will perform studies in large animal models, including rabbits and pigs, to optimize drug loading according to body weight or defect size. These studies will include long-term evaluations to assess complete bone regeneration. Additionally, the choice or combination of drugs in COHAS may need adjustment depending on the specific bone defect for optimal efficacy. 5. Conclusion We fabricated COHAS and developed a sequential drug release system that co-loads osteoblast- stimulating BMP-2 and osteoclast-inhibiting OPG-Fc. We confirmed that BMP-2 enhances osteogenic activity in osteoblasts, while OPG-Fc effectively inhibits preosteoclast differentiation in vitro. Inspired by the natural bone healing process, in which bone resorption is orchestrated by prolonged osteoclast differentiation and activation, we encapsulated OPG-Fc within PLGA microspheres for sustained and delayed release. This approach produced a synergistic bone healing effect when combined with BMP-2. Our results demonstrate that COHAS_B_P@O effectively promotes bone healing by activating osteoblasts throughout bone regeneration and inhibiting osteoclast precursor differentiation from the intermediate stage. Also, all COHAS formulations co-loaded with drugs showed no cytotoxicity and exhibited good biocompatibility, which are essential for biomaterial utilization. We believe that our dual-drug delivery platform of BMP-2 and OPG-Fc can contribute to harnessing the potential of sequential drug release scaffolds for bone regeneration. Declarations Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Funding This work was supported by the National Research Foundation of Korea (NRF) grants from the Korean Government (MSIP) (Grant Nos. 2022R1A6A3A13072828, 2022R1A2C1091756, RS-2024-00400563, RS-2024-00414209, 2018M3C1B7020716, 2018M3C1B7020722, 2019M3C1B7026601). Amgen Inc provided the OPG-Fc under a research program agreement (No. 10655700). This work was supported by BK21 FOUR Institute of Precision Public Health. This work was also supported from Hyundai Motor Chung Mong-Koo Foundation. Author Contribution Author ContributionsJ.W.J., H.K., and J.S.O. contributed equally to this work. H.S., C.N.S., S.H.K., H.K., and D.S.Y. performed funding acquisition, project administration, supervision. J.W.J and H.K. fabricated the drug-loaded scaffolds. J.W.J., H.K., D.L., H.G.J., Y.H.K. and J.S.Y. characterized the drug-loaded scaffolds and analyzed the data. H.J.C. and C.N.S. provided the cell lines. J.S.O., and X.J. performed the animal experiments and analyzed the data. J.W.J., H.K., and J.S.O. co-wrote the original draft. D.L. and D.S.Y. reviewed and edited the draft. Data Availability The data that support the findings of this study are available within the article and its supplementary information files, and are also available from the corresponding author upon reasonable request. AUTHOR INFORMATION Corresponding Author Dae Sung Yoon - School of Biomedical Engineering, Korea University, Seoul 02841, South Korea; Interdisciplinary Program in Precision Public Health, Korea University, Seoul 02841, South Korea; ASTRION, 47, Gaeunsa-gil, Seongbuk-gu, Seoul 02842, Republic of Korea; Email: [email protected] References Bouvard, B., Annweiler, C. & Legrand, E. Osteoporosis in older adults. Joint Bone Spine . 88 , 105135 (2021). Pignolo, R. J., Law, S. F. & Chandra, A. Bone aging, cellular senescence, and osteoporosis. JBMR plus . 5 , e10488 (2021). Durbano, H. W. et al. Aberrant BMP2 signaling in patients diagnosed with osteoporosis. Int. J. Mol. 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06:53:17","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":527795,"visible":true,"origin":"","legend":"\u003cp\u003eScheme of (a) COHAS_B_P@O synthesis, (b) mechanism of BMP-2 and P@O, and (c) in vivo experiment of scaffold implantation in rat model.\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-7438279/v1/993d3651052f6a2142ac80fa.png"},{"id":91592147,"identity":"d3a49f13-84b5-4b18-a7d2-337df82d8bef","added_by":"auto","created_at":"2025-09-18 06:53:18","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":443753,"visible":true,"origin":"","legend":"\u003cp\u003eScanning electron microscopic images of (a) COHAS, (b) P@O, and (c) COHAS_B_P@O.\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-7438279/v1/b0373589b206fbd66b0f2190.png"},{"id":91593824,"identity":"fd9fa0d5-aa92-4ec4-ba41-d1f757715568","added_by":"auto","created_at":"2025-09-18 07:09:18","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":211488,"visible":true,"origin":"","legend":"\u003cp\u003eDrug release ratios of OPG-Fc depending on the encapsulation of PLGA microspheres in the COHAS.\u003cstrong\u003e (a) \u003c/strong\u003eSchematic illustration of OPG-Fc release in direct and delayed form.\u003cstrong\u003e (b) \u003c/strong\u003epreosteoclast differentiation inhibition\u003cstrong\u003e \u003c/strong\u003eby OPG-Fc. The amount of treated sRANKL was 100 ng. \u003cstrong\u003e(c)\u003c/strong\u003e OPG-Fc release kinetics of COHAS_O and COHAS_P@O. \u003cstrong\u003e(d)\u003c/strong\u003e BMP-2 release kinetics of COHAS_B and COHAS_P@B. Every individual data point represents a set of triplicate measurements. Every individual data point represents a set of triplicate measurements.\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-7438279/v1/4283d9bd3ebe14a3a64c56c7.png"},{"id":91592148,"identity":"170ff360-e5ae-4912-9a72-8a1c21587d95","added_by":"auto","created_at":"2025-09-18 06:53:18","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":53451,"visible":true,"origin":"","legend":"\u003cp\u003eIn vitro assay of (a) cell viability and (b) ALP activity with different groups of COHAS. Every individual data point represents a set of triplicate measurements.\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-7438279/v1/1ba24a841d609533ac2fade8.png"},{"id":91592142,"identity":"00a9f105-bec3-4c5f-92fb-cf1f92ab9f56","added_by":"auto","created_at":"2025-09-18 06:53:17","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":735400,"visible":true,"origin":"","legend":"\u003cp\u003eIn vivo analysis of different COHAS groups: COHAS, COHAS_B, COHAS_O, COHAS_B_O, COHAS_P@B_O, COHAS_B_P@O. (a) Micro-computed tomography (µ-CT) images of each group at 2, 4, and 8 weeks following the surgical procedure. Native bone is shown in red, regenerated bone in green, and unmineralized defect areas in black. Scale bar represents 1 mm. (b) Absolute regenerated bone area and (c) absolute regenerated bone volume from µ-CT analysis. Every individual data point represents a set of triplicate measurements. Statistical significance is indicated as follows: ** (p \u0026lt; 0.01), *** (p \u0026lt; 0.001), **** (p \u0026lt; 0.0001).\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-7438279/v1/5cb54ae5496878a7e76e6ac4.png"},{"id":91592146,"identity":"2d8ed71f-1d08-4ba9-875d-18d2283f9069","added_by":"auto","created_at":"2025-09-18 06:53:17","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":775399,"visible":true,"origin":"","legend":"\u003cp\u003eHistological evaluation of bone regeneration of calvarial defects in 8 weeks post implantation of various groups of COHAS: COHAS, COHAS_B, COHAS_O, COHAS_B_O, COHAS_P@B_O, COHAS_B_P@O. (a) Hematoxylin and eosin (H\u0026amp;E) and Masson's trichrome (MT) stain image. Non-mineralized osteoid connective tissue is shown in blue, while mineralized bone appears red. IC: Inflammatory cell, LB: Lamellar bone, FT: fibrous tissue, CB: Cortical bone (×10 and ×400 magnification, scale bar = 3 mm and 50 um each). (b) Semi-quantitative analysis of red stained area ratio (%) of MT staining in 8 weeks. Every individual data point represents a set of triplicate measurements. Statistical significance is indicated as follows: * (p \u0026lt; 0.05)\u003c/p\u003e","description":"","filename":"image6.png","url":"https://assets-eu.researchsquare.com/files/rs-7438279/v1/756e64957ef2ef72fc07dc4c.png"},{"id":92600339,"identity":"a8e94f59-6632-4dc8-a4e3-7832f8391be2","added_by":"auto","created_at":"2025-10-01 14:21:14","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3564275,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7438279/v1/0e867e6e-c2f0-4162-85d9-cd8cd0f343df.pdf"},{"id":91592152,"identity":"d3c243f9-1eb9-4b77-9396-fa14bb6f7756","added_by":"auto","created_at":"2025-09-18 06:53:18","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":9197069,"visible":true,"origin":"","legend":"","description":"","filename":"Supplementarymaterial.docx","url":"https://assets-eu.researchsquare.com/files/rs-7438279/v1/a9ae4aab5ee6f840257d5039.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Synergistic Bone Regeneration Through Sequential Dual-drug Delivery","fulltext":[{"header":"1. INTRODUCTION","content":"\u003cp\u003eAs the global population continues to age, there is an increasing concern about age-related health issues such as osteoporosis and bone fractures\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. Osteoporosis is a condition that weakens bones, making them fragile and prone to fractures. Fractures resulting from osteoporosis most often occur in the hip, spine, and wrist and can have serious consequences, including disability and even death\u003csup\u003e\u003cspan additionalcitationids=\"CR4\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. Also, the large bone defects stemming from osteosarcoma, infections, and injuries from physical trauma should also be considered, which all demand a challenging process for treatment\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. The conventional treatment methods, including autograft, allograft, and xenograft, have some drawbacks like limited obtainable quantities, immunogenic graft rejection, and nonunion of graft-host junction\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. Furthermore, the cell-based bone repair method using stem cells requires prolonged time for healing and a high price\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. To overcome these limitations, various strategies involving bioactive molecules and biomaterials are being explored to enhance bone regeneration by creating an optimal microenvironment that supports cell migration, proliferation, and differentiation\u003csup\u003e\u003cspan additionalcitationids=\"CR10\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eFor bone tissue regeneration, a three-dimensional scaffold with a porous architecture plays a crucial role by providing a supportive environment that accommodates preosteoblasts or bone marrow mesenchymal stem cells and promotes their differentiation into mature cells.\u003csup\u003e\u003cspan additionalcitationids=\"CR13 CR14 CR15\" citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. Collagen and hydroxyapatite, the main components of natural bone, are widely used biomaterials for bone regeneration scaffolds\u003csup\u003e\u003cspan additionalcitationids=\"CR18\" citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e. Their availability and versatility enable the the mass production of bone scaffolds, offering a promising alternative to existing bone graft materials\u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. Collagen shows excellent cell adhesion properties by providing numerous binding sites for cell membrane receptors, which contribute to high osteoinductivity\u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. However, substrates composed solely of collagen have poor mechanical strength, rendering them insufficient to maintain structural integrity at bone defect sites\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. Hydroxyapatite, a well-known osteoconductive biomaterial, can be incorporated to enhance structural support by increasing mechanical strength and to promote bone regeneration by releasing bone-forming ions and bioactive molecules in a controlled manner\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eBone morphogenetic protein-2 (BMP-2), an osteoinductive growth factor approved by the Food and Drug Administration (FDA), is widely recognized as a key biological agent encapsulated within scaffolds for bone regeneration\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. BMP-2 is produced by osteoblasts and plays a critical role in multiple stages of bone formation, including osteoblast proliferation and differentiation, bone matrix production and mineralization, and bone tissue remodeling\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. However, its high dose and uncontrolled release can lead to potential complications, including heterotopic ossification, tissue inflammation, and the induction of osteoclastogenesis multiple molecular pathways\u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e,\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e,\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. Therefore, careful optimization of dosing and delivery methods is essential to maximize the therapeutic benefits of BMP-2 while minimizing its associated risks. Osteoprotegerin (OPG), another FDA-approved biological agent used in bone regeneration, is an innate protein that regulates bone metabolism by inhibiting the receptor activator of nuclear factor-κB ligand (RANKL)\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. In particular, the Fc region is added to OPG to create OPG-Fc, which extends the protein\u0026rsquo;s half-life\u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. OPG-Fc promotes new bone formation by suppressing osteoclast differentiation and activity. Both biological agents have demonstrated effectiveness in promoting bone formation in preclinical and clinical studies involving various bone disorders, including osteoporosis, bone metastasis, and fractures\u003csup\u003e\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e,\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. Previous studies have explored the combined use of BMP-2 and OPG to enhance the bone healing process. For instance, Bougioukli et al. demonstrated advancements in new bone formation and integration at the site of a mouse critical-sized femoral defect by delivering BMP-2 and OPG-Fc\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. OPG-Fc was systemically administered through subcutaneous injection. A notable limitation of this approach is the absence of a drug carrier for OPG-Fc delivery, which could potentially lead to osteopetrosis due to elevated drug absorption. Wang et al. showed improved osseointegration in an osteoporotic animal model by employing 3D-printed composite scaffolds loaded with BMP-2 and OPG\u003csup\u003e\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e. Wei et al. utilized a collagen sponge patch loaded with BMP-2 and OPG in a rabbit joint surgery model, leading to enhanced tendon-bone healing\u003csup\u003e\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e\u003c/sup\u003e. Both studies confirmed the sustained release of BMP-2 and OPG; however, they were limited to simultaneous release only and lacked a mechanism for sequential, delayed release that reflects the entire bone regeneration process.\u003c/p\u003e\u003cp\u003eIn the natural bone healing process, the initial stage involves the differentiation of the osteoclast precursors following the differentiation and activation of osteoblasts\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. The osteoclast cycle begins approximately five days after the onset of the bone defect. During this cycle, osteoclasts resorb bone by secreting hydrogen ions and proteolytic enzymes into the local microenvironment. This process typically lasts for about two to three weeks. The activation of osteoclasts during the early stage helps remove debris and residual material from the site of bone damage, creating an optimal environment for bone formation. However, if osteoclasts remain persistently active beyond this stage, they may impede bone formation. Therefore, inhibiting osteoclastogenesis after the early stage is necessary to enhance the effectiveness of bone regeneration. In this context, the simultaneous release of a bone growth factor and osteoclastogenesis inhibitor may reduce the efficacy of new bone formation. The inhibitor may unnecessarily suppress osteoclast activity during the early stage, while its short half-life limits its effectiveness during the intermediate stage. Accordingly, we propose that sequential delivery\u0026mdash;specifically, delayed release of the osteoclastogenesis inhibitor following the bone growth factor\u0026mdash;represents a promising strategy for enhancing bone regeneration. The delayed release of osteoclastogenesis inhibitor is thought to prevent unnecessary inhibition during the early stage. In this regard, Lee et al. demonstrated that sequential release of the BMP-2 and alendronate (i.e., delayed release of alendronate) can significantly enhance new bone formation\u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. Alendronate is a small-molecule osteoclast inhibitor and a common therapeutic agent for osteoporosis. Building on these findings, we speculate that delayed release of biological agents such as antibodies (e.g., OPG-Fc) can also effectively inhibit osteoclast activity after the early stage, leading to more efficient bone regeneration. Investigating the clinical impact of delayed release of biological inhibitors on bone regeneration is therefore important. However, studies dealing with this strategy\u0026mdash;particularly, the delayed release of antibodies\u0026mdash;have yet to be published.\u003c/p\u003e\u003cp\u003eIn our study, we engineered a cell-free bone regeneration scaffold, the collagen-hydroxyapatite scaffold (COHAS), incorporating BMP-2 and OPG-Fc to enhance bone regeneration (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). This scaffold mimics natural bone and serves as an alternative to bone grafts, offering excellent osteoconductivity. It also acts as a reservoir for two factors: BMP-2 and OPG-Fc (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea). We hypothesized that sequential release of BMP-2 and OPG-Fc would have a synergistic effect on bone regeneration. The initial release of BMP-2 triggers osteogenic differentiation of mesenchymal stem cells (MSCs), promoting osteogenesis. Subsequently, OPG-Fc is released to inhibit osteoclast differentiation by binding to RANKL, thereby further enhancing bone regeneration (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eb). We assessed the \u003cem\u003ein vitro\u003c/em\u003e and \u003cem\u003ein vivo\u003c/em\u003e effects of this sequential release and compared them with single or simultaneous release of BMP-2 and OPG-Fc, as summarized in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. Our results demonstrated that sequential release significantly improved bone regeneration with minimal inflammation at 8 week post-implantation in an 8 mm calvarial defect rat model (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ec).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e"},{"header":"2. MATERIALS AND METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1 Materials\u003c/h2\u003e\u003cp\u003eDulbecco\u0026rsquo;s modified eagle\u0026rsquo;s medium (DMEM) (Corning, USA), fetal bovine serum (Welgene, South Korea) and penicillin-streptomycin (Corning, USA) were used to culture preosteoclast cell. Alpha-MEM (α-MEM) (Welgene, South Korea), fetal bovine serum and penicillin-streptomycin were used to culture preosteoblast cell. We purchased soluble Receptor Activator of NFkB Ligand (sRANKL) from PROSPEC Protein Speciallists (USA), recombinant human bone morphogenetic protein-2 (rhBMP-2) from Peprotech (USA), carboxyl-terminated 50:50 PLGA (0.67 dl/g) from LACTEL Absorbable Polymers (USA), collagen Type 1 Rat Tail of 3.8 mg/mL from Corning (USA) and hydroxyapatite nanopowder(\u0026lt;\u0026thinsp;200nm particle size) from Sigma Aldrich (USA). Human Osteoprotegrin/TNFRSF11B Duoset ELISA and human BMP-2 ELISA kit were purchased from R\u0026amp;D systems (USA) and ABclonal (USA). The cell counting-8 kit (CCK-8) was purchased from Merck KGaA (Germany). Tartrate-resistant acid phosphatase assay (TRACP) kit and alkaline phosphatase assay (ALP) kit was purchased from Takara Bio (Japan). Preosteoblast MC3T3-E1 cells, human osteoprotegerin (OPG-Fc) were provided by Eulji Medical Center.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2 Fabrication of collagen-hydroxyapatite scaffold\u003c/h2\u003e\u003cp\u003eA collagen-hydroxyapatite scaffold was fabricated following a previous method with minor modification\u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. Briefly, COHAS slurry was prepared by incorporating 100 mg hydroxyapatite powder into the 2 ml collagen matrix. All procedures were carried out at a temperature of 4\u0026deg;C. To ensure a homogeneous mixture, the slurry was subjected to high-speed homogenization at a minimum of 10,000 rpm for a duration of 5 minutes. Subsequently, a degassing process was employed to eliminate any undesired bubbles. The degassed slurry was then combined with a neutralization solution within an 8 mm diameter polydimethylsiloxane (PDMS) mold and stirred. The resulting mixture was incubated at 37\u0026deg;C for 1 hour and subsequently transferred to a deep-freezer set at -80\u0026deg;C. The frozen scaffold was subjected to freeze-drying to obtain the final COHAS. Prior to use, the scaffold was sterilized using UV light (λ\u0026thinsp;=\u0026thinsp;254 nm) for a duration of 4 hours.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\u003ch2\u003e2.3 Mechanical property characterization of collagen-hydroxyapatite scaffold\u003c/h2\u003e\u003cp\u003eCompressive strength testing was conducted using a universal testing machine (UTM; ST-1000, Salt Co., Ltd., Incheon, Republic of Korea). Cylindrical scaffold specimens, prepared in the swollen state after immersion in phosphate-buffered saline (PBS), had an average diameter of approximately 8 mm and a height of approximately 5 mm. Three distinct specimens were tested for statistical reliability. All tests were performed under displacement control at a crosshead speed of 1 mm/min. A preload of 0.1 N was applied before measurement, and the compressive stress\u0026ndash;strain curves were obtained from the recorded load\u0026ndash;displacement data. Stress was calculated by normalizing the compressive load to the initial cross-sectional area, and strain was determined from the change in specimen height relative to the initial height. The compressive strength was defined as the average stress within the plateau region, corresponding to a strain range of 10\u0026ndash;30%.\u003c/p\u003e\u003cp\u003eThe porosity of the freeze-dried collagen-hydroxyapatite scaffolds was determined from the measured scaffold density and the theoretical composite density. The mass of each dried scaffold (n\u0026thinsp;=\u0026thinsp;8) was measured using a digital balance (PioneerTM, OHAUS Co., USA; accuracy: \u0026plusmn; 0.0001 g), and their volume was determined from its dimensions obtained using a digital Vernier caliper (CD-15APX, Mitutoyo Co., Japan; accuracy: \u0026plusmn; 0.01 mm). The apparent scaffold density (ρ_scaffold) was calculated as:\u003cdiv id=\"Equa\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equa\" name=\"EquationSource\"\u003e\n$$\\:{\\rho\\:}_{scaffold}=\\frac{{m}_{scaffold}}{{V}_{scaffold}}$$\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e\u003cp\u003ewhere \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{m}_{scaffold}\\)\u003c/span\u003e\u003c/span\u003e is the dried scaffold mass and \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:{V}_{scaffold}\\)\u003c/span\u003e\u003c/span\u003e​ is the volume of scaffold.\u003c/p\u003e\u003cp\u003eThe theoretical density of the solid composite \u003cspan class=\"InlineEquation\"\u003e\u003cspan class=\"mathinline\"\u003e\\(\\:\\left({\\rho\\:}_{material}\\right)\\)\u003c/span\u003e\u003c/span\u003e was calculated from the masses of collagen and hydroxyapatite used and their theoretical densities, assumed to be 1.23 g/cm\u0026sup3; and 3.14 g/cm\u0026sup3;, respectively. The percent porosity (%) was then determined as,\u003cdiv id=\"Equb\" class=\"Equation\"\u003e\u003cdiv format=\"TEX\" class=\"mathdisplay\" id=\"FileID_Equb\" name=\"EquationSource\"\u003e\n$$\\:Porosity\\:\\left(\\%\\right)=\\left(1-\\:\\frac{{\\rho\\:}_{scaffold}}{{\\rho\\:}_{material}}\\:\\right)\\:\\times\\:100$$\u003c/div\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\u003ch2\u003e2.4 Synthesis of PLGA microsphere containing drugs\u003c/h2\u003e\u003cp\u003ePLGA microsphere was synthesized using oil-in-water (O/W) technique\u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. To prepare the PLGA-based formulation, 100 mg of PLGA polymer was dissolved in 2 ml of dichloromethane through stirring. rhBMP-2 and OPG-Fc were separately added to the PLGA solution. The resulting PLGA solution was then introduced dropwise into a stirring 0.5% polyvinyl alcohol (PVA) solution. The mixed solution was homogenized at 3,000 rpm for 30 seconds. The emulsion obtained from the homogenization step was stirred using magnetic stirrer to facilitate vaporization of organic solvent. Subsequently, the mixture was transferred to a conical tube and centrifuged at 3,000 rpm for 3 minutes. The supernatant was carefully collected and washed 3 times with distilled water.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section2\"\u003e\u003ch2\u003e2.5 Drug release kinetics of OPG-Fc\u003c/h2\u003e\u003cp\u003eThe drug release kinetics of OPG-Fc was evaluated by a reported method\u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. In brief, the COHAS samples were placed in separate wells of a 6-well plate. The total amount of OPG-Fc was 10 \u0026micro;g in each sample. Each well contained 2 ml of PBS. The plate was then subjected to shaking at 37\u0026deg;C for the duration of the incubation period (WIS-20, Daihan Scientific, Korea). The solution was extracted from the 6-well plate at given time points and re-filled with same amount of PBS. The release profile was examined by OPG-Fc ELISA (DuoSet ELISA, R\u0026amp;D systems, USA) according to the protocol provided from manufacturer via a microplate reader (SpectraMax ABS Plus, Molecular Devices, CA, USA). The standard graph was determined using the microplate reader set to 450 nm. Release kinetic of OPG-Fc was quantified by the same procedure.\u003c/p\u003e\u003cp\u003eIn addition, we defined the apparent encapsulation efficiency (EE_app) as the cumulative amount released at the plateau (day 56) divided by the initial input (10 \u0026micro;g) \u0026times; 100. We note that this EE_app represents a conservative lower-bound estimate of the true encapsulation efficiency because incomplete release and potential analytical loss may lead to underestimation of the encapsulated amount.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003e2.6 Drug release kinetics of BMP-2\u003c/h2\u003e\u003cp\u003eThe drug release kinetics of BMP-2 was evaluated by a reported method\u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. The hydrogels were placed individually in 6-well plate with 2 ml of PBS. 1 \u0026micro;g of BMP-2 was contained in each hydrogel and the hydrogels were incubated in 37\u0026deg;C shaking incubator (WIS-20, Daihan Scientific, Korea). We extracted the solution at fixed times and replaced it with fresh PBS. The drug concentration was examined by human BMP-2 ELISA kit (ABclonal, MA, USA) by provided protocol. The release kinetics and the standard graph were measured using the microplate reader set to 450 nm and the wavelengths were corrected with 630 nm for optical imperfections.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec9\" class=\"Section2\"\u003e\u003ch2\u003e2.7 In vitro Tests\u003c/h2\u003e\u003cp\u003eThe preosteoclast RAW 264.7 cells were cultured in DMEM supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin. The cell culture was maintained in a 5% CO\u003csub\u003e2\u003c/sub\u003e atmosphere at 37\u0026deg;C. TRACP assay kit was performed to confirm the osteoclast differentiation inhibitory effect of OPG-Fc. 5 x 10\u003csup\u003e3\u003c/sup\u003e cells were incubated in each well of 96-well plate and 50 \u0026micro;g/ml of sRANKL was contained to promote osteoclast differentiation. The 200 \u0026micro;l of media was added for cell culture and changed with appropriate medium every 2 days. The cell differentiation was examined at 1, 3, 7 days and the solution was diluted 20-folded for measurement. The absorbance of groups was measured with microplate reader.\u003c/p\u003e\u003cp\u003eThe preosteoblast MC3T3-E1 cells were cultured in alpha-minimum essential medium (α-MEM) supplemented with 10% fetal bovine serum and 1% penicillin-streptomycin in a 5% CO\u003csub\u003e2\u003c/sub\u003e at 37\u0026deg;C. To assess cell proliferation, a Cell Counting Kit-8 (CCK-8) assay was performed following the manufacturer's protocol. Briefly, the MC3T3-E1 cells were seeded at a density of 5 x 10\u003csup\u003e3\u003c/sup\u003e cells per well in a 96-well plate containing COHASs incorporated with 1 \u0026micro;g of rhBMP-2 and 10 \u0026micro;g of OPG-Fc. The cell viability was examined using a microplate reader at days 1, 3, and 7. The cell culture medium in each well was changed every two days. The differentiation of cells was evaluated via ALP assay at 1, 3, and 7 days. The intensity of the resultant color was measured in a dark setting to accurately quantify the ALP activity.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003e2.8 In vivo study\u003c/h2\u003e\u003cp\u003e All animal experiments were approved by the Institutional Animal Care and Use Committee in Seoul National University Hospital (SNUH-IACUC) with IACUC No. 21-0202-S1A) and animals were maintained in the facility accredited AAALAC International (#001169) in accordance with Guide for the Care and Use of Laboratory Animals 8th edition, NRC (2010). Eighteen healthy male Sprague-Dawley rats (2 months old, weighing 250\u0026ndash;300 g) supplied by Koatech, Keung-Ki, Pyong-Taek, Korea, were used for the study. Each rat was assigned a unique ID and randomly allocated into one of six experimental groups (n\u0026thinsp;=\u0026thinsp;3 per group). The scaffold was fabricated with 1 \u0026micro;g of BMP-2 and 50 \u0026micro;g of OPG-Fc for in vivo experiment.\u003c/p\u003e\u003cp\u003eWe selected animals with similar age, sex, and body weight for the experiments and assigned each animal a unique ID before randomly allocating them into experimental groups. The surgeon was aware of the group assignments during the procedure; however, the outcome evaluator was blinded to group allocation and assessed the results based solely on the animal IDs. This blinding approach minimized potential bias and ensured the objectivity of the outcome assessment.\u003c/p\u003e\u003cp\u003eFor critical size calvarial defect model, the animals were anesthetized by using 2\u0026ndash;5% isoflurane for induction and 0.25\u0026thinsp;~\u0026thinsp;0.4% for maintenance. Their scalp was shaved from the end of the skull to the midpoint between the eyes using electric clippers. Betadine (Hyundai Pharm, Korea) were used for disinfection. With a no.15 blade, an incision sized 4 cm was made down to bone level. Periosteum was dissected laterally to expose the underlying skull using an elevator. Trephination using 8 mm-sized electrical burr (Seoul, South Korea) was done to make the bone defect. Periosteum was closed with running 4\u0026thinsp;\u0026minus;\u0026thinsp;0 Monocryl suture. Skin closure was done using running suture with 4\u0026thinsp;\u0026minus;\u0026thinsp;0 Prolene.\u003c/p\u003e\u003cp\u003eBone regeneration was evaluated using in vivo micro computed topography (\u0026micro;-CT; NFR Polaris-G90, Nano Focus Ray, Korea) with isotropic voxel size of 9 \u0026micro;m and X-ray tube voltage at 55kV and current at 180 \u0026micro;A. Imaging was done under anesthesia using isoflurane at 2, 4, and 8 weeks after COHAS insertion. At each point the regenerated bone volume and surface area were evaluated and calculated using Analyze (Version 12.0, AnalyzeDirect, Inc. USA). For histopathological examinations, hematoxylins-eosin (H\u0026amp;E) and Masson\u0026rsquo;s trichrome (MT) staining methods were conducted. The skull tissue encompassing both the defect and the surrounding normal bone was collected after an 8-week period. Subsequently, the tissue samples were fixed in 10% buffered formalin for 3 days, decalcified for 7 days, and sectioned into 5 \u0026micro;m thick slices. The samples were examined under a light microscope (BX53F, OLYMPUS, Japan) photographed at \u0026times;40, \u0026times;100, and \u0026times;400 magnifications.\u003c/p\u003e\u003cp\u003eThis study was reported in accordance with the ARRIVE guidelines.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003e2.9 Statistical Analysis\u003c/h2\u003e\u003cp\u003eAll statistical analysis was performed by the Medical Research Collaborating Center at Seoul National University Hospital using SAS statistical software (SAS system for Windows, version 9.3; SAS Institute, Cary, NC, USA). A mixed model analysis was performed to test for differences in bone formation area according to drug combinations, accounting for repeated time measurements. The interaction between drug combination and time point was found to be non-significant and was therefore removed from the model. At the 8-week time point, a mixed model analysis including the group-time interaction term was conducted to examine differences between COHAS and COHAS_B_P@O, and between COHAS_B_P and COHAS_B_P@O, for bone regeneration area and volume. A linear mixed model was used to account for variance from repeated measurements over time.\u003c/p\u003e\u003cp\u003eMultiple group comparisons were performed without correction for increased Type I error, due to the exploratory nature of this animal study. Although up to 15 pairwise comparisons were possible among the six groups (6C2\u0026thinsp;=\u0026thinsp;15), the main comparisons of interest were predefined. For transparency, exact p-values and 95% confidence intervals are reported in Supplementary Tables S2\u0026ndash;S6. Significant statistical differences were indicated as *p\u0026thinsp;\u0026lt;\u0026thinsp;0.05, **p\u0026thinsp;\u0026lt;\u0026thinsp;0.01, ***p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, and ****p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001.\u003c/p\u003e\u003c/div\u003e"},{"header":"3. RESULTS","content":"\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003e3.1. Morphological Analysis and Mechanical Property Characterization of Scaffold\u003c/h2\u003e\u003cp\u003eThe morphology and dimensions of the hydroxyapatite-based scaffold were examined using scanning electron microscopy (SEM). To achieve sequential delivery of BMP-2 and OPG-Fc, we fabricated PLGA microspheres and a drug-loading scaffold based on our previous study. The scaffold, COHAS, consisted of collagen and hydroxyapatite. Their ratio was carefully optimized to support effective drug delivery. Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e lists the abbreviations for scaffold samples with different combinations of BMP-2, OPG-Fc, and PLGA microspheres. The cross-section of the lyophilized collagen hydrogel showed a porous structure with a large surface area, suitable for accommodating hydroxyapatite (Figure \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e, Supporting Information). Compared to scaffolds made solely of hydroxyapatite or collagen, COHAS displayed a distinct, more intricate structure, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ea. The average size of the PLGA microspheres containing OPG-Fc (P@O) was approximately 100 \u0026micro;m, indicating a microscopic scale (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eb). These microspheres possess a porous structure that allows drug loading and can be modified to control drug release\u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. Figure\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003ec shows the inner section of COHAS_B_P@O, where the collagen matrix and PLGA microspheres coexist. Since OPG-Fc is encapsulated within the PLGA microspheres dispersed in COHAS, it must pass through two barriers before reaching the external environment. Thus, in COHAS_B_P@O, OPG-Fc release follows the release of BMP-2, ensuring sequential delivery.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eThe compressive strength of the COHAS was 2.80\u0026thinsp;\u0026plusmn;\u0026thinsp;0.17 kPa calculated from Figure S2. This value is within the typical range for COHASs used in bone tissue engineering. Many works note that optimal osteoblast proliferation and maturation can happen on matrices softer than bone itself, often below 10 kPa\u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e,\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. Some evidence indicates that softer scaffolds (~\u0026thinsp;1\u0026ndash;3 kPa) can even enhance osteogenic differentiation at early stages compared to harder substrates\u003csup\u003e\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u003c/sup\u003e. Therefore, these previous reports support that the compressive strength of our scaffold is suitable for bone repair, particularly during the initial phases of bone healing.\u003c/p\u003e\u003cp\u003eThe percent porosity of the scaffold was 95.82% which measured from eight COHASs, using the equation (Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e). Porosity above 90% is often targeted in bone scaffolds because it provides a large surface area and interconnected pore network that facilitates cell adhesion, migration, nutrient and oxygen diffusion, and vascularization. These features support robust bone tissue regeneration\u003csup\u003e\u003cspan additionalcitationids=\"CR42\" citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e. Therefore, the high porosity of our scaffold is considered advantageous for bone tissue regeneration.\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eAbbreviations for the various samples used in the study.\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"2\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eName of sample\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eDescription of sample\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCOHAS\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003ecollagen-hydroxyapatite scaffold\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eP@O\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003ePLGA microspheres containing OPG-Fc\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCOHAS_B\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003escaffold incorporated with BMP-2\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCOHAS_O\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003escaffold incorporated with OPG-Fc\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCOHAS_B_O\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003escaffold incorporated with BMP-2 and OPG-Fc\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCOHAS_P@B_O\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003escaffold incorporated with BMP-2 encapsulated in PLGA microspheres and OPG-Fc\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003eCOHAS_B_P@O\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c2\"\u003e\u003cp\u003escaffold incorporated with BMP-2 and OPG-Fc encapsulated in PLGA microspheres\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003c/table\u003e\u003c/div\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003e3.2. In Vitro Release of OPG-Fc and BMP-2 from COHAS\u003c/h2\u003e\u003cp\u003eAccurately monitoring the concentration of bioactive molecules in vivo is nearly impossible. Therefore, we investigated the release patterns of BMP-2 and OPG-Fc from the scaffolds in vitro by simulating body fluid conditions. The Scaffolds were immersed in phosphate-buffered saline (PBS), which has ionic concentration and osmotic pressure similar to human body fluid and helps maintain stable pH. We quantified OPG-Fc release from two COHAS groups, COHAS_O and COHAS_P@O, using OPG-Fc ELISA kits (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ea).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eCOHAS_O showed burst release behavior, with a significant amount of OPG-Fc released by day 8. In contrast, COHAS_P@O exhibited a delayed release profile. It steadily released the drug until day 42 and reached saturation by day 56 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec). This delayed release is due to the encapsulation of OPG-Fc within PLGA microspheres. These microspheres slow the diffusion of the drug from COHAS to the external environment. The cumulative amounts of OPG-Fc released from COHAS_O and COHAS_P@O differed by only 7.13% at day 56. This indicates that OPG-Fc was effectively encapsulated in the PLGA microspheres and demonstrated the stable release performance. The loading efficiency of OPG-Fc was approximately 88.2% for COHAS_O and 81.9% for COHAS_P@O, based on the released amount relative to the initial loading. These results indicate a notably higher drug loading rate compared to previous studies\u003csup\u003e\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e. They also suggest that OPG-Fc has minimal interaction with either COHAS or PLGA.\u003c/p\u003e\u003cp\u003eThe inhibitory effect of OPG-Fc on preosteoclast differentiation, specifically osteoclastogenesis, was tested by adding varying concentrations of OPG-Fc to RAW264.7 cells in the presence of the osteoclast differentiation factor, soluble RANKL (sRANKL), as depicted in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eb. Osteoclastic differentiation was measured using the tartrate-resistant acid phosphatase assay (TRACP) assay kit. Compared to the sRANKL-only group, all OPG-Fc treated groups (10, 50, 100 \u0026micro;g) showed consistent inhibition on both day 3 and day 7. Notably, on day 7, the inhibition rates increased with OPG-Fc concentration, reaching 84.0%, 93.0%, and 97.2% for 10 \u0026micro;g, 50 \u0026micro;g, and 100 \u0026micro;g, respectively.\u003c/p\u003e\u003cp\u003eThe BMP-2 release profile of COHAS_B was measured using BMP-2 ELISA kits (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed). The release patterns of BMP-2 from both COHAS_B and the COHAS_P@B were similar to those shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec. Specifically, COHAS_B exhibited a burst release, with about 50% of the total BMP-2 released on day 1 and approximately 72% by day 14. In contrast, COHAS_P@B showed a delayed release, reaching around 73% release only by day 42. This delay is attributed to BMP-2 encapsulation within PLGA microspheres.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec15\" class=\"Section2\"\u003e\u003ch2\u003e3.3. In Vitro Cell Proliferation \u0026amp; Osteogenesis study\u003c/h2\u003e\u003cp\u003eTo assess the cytotoxic effects of various COHAS groups, a cell viability assay was performed using the cell counting kit-8 (CCK-8) on days 1 and 7. The control consisted of cells incubated without COHAS. On day 7, all COHAS groups were confirmed to be non-toxic and did not affect cell growth (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea). This finding also suggests that both bioactive agents, BMP-2 and OPG-Fc, have excellent biocompatibility (Figure S3a, Supporting Information). The osteogenic effects of the COHAS groups were evaluated via alkaline phosphatase (ALP) assay kits. The ALP activity of COHAS_O on day 7 exhibited minimal change compared to day 1, indicating that OPG-Fc alone is insufficient to activate preosteoblasts (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb). This is supported by data in Figure S3b, which demonstrates that preosteoblasts did not differentiate or activate when directly exposed to OPG-Fc. COHAS_B_O showed a greater increase in ALP activity compared to COHAS_B, suggesting a potential synergistic effect from the simultaneous application of BMP-2 and OPG-Fc. Moreover, COHAS_B_P@O demonstrated the highest ALP activity among all groups. This is likely due to the delayed release of OPG-Fc and its synergistic interaction with BMP-2 (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section2\"\u003e\u003ch2\u003e3.4. In Vivo Study\u003c/h2\u003e\u003cp\u003eTo assess bone regeneration, a critical-size calvarial defect rat model was used. A preformed COHAS was carefully inserted into the defect area, as shown in Figure S4. The animals were divided into six groups, each consisting of three rats. The calvarial defects were filled with different materials: COHAS, COHAS_B, COHAS_O, COHAS_B_O, COHAS_B@P_O, and COHAS_B_P@O. Figure\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea presents 3D reconstructed image illustrating bone regeneration in all groups. Bone growth was observed in both the central and peripheral regions of the defect site. The average area and volume of regenerated bone for each group are summarized in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb, Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ec, and Table S2. The results revealed clear variations in bone regeneration among the groups. COHAS had the smallest new bone area and volume, while COHAS_B_P@O demonstrated the highest level of regeneration. Tables S3 and S4 provide a comprehensive comparison of area and volume measurements, using COHAS as the reference. Importantly, COHAS_P@B_O exhibited a modest enhancement in bone growth compared to COHAS_B_P@O. This highlights the significant impact of drug release sequence on bone regeneration efficiency. Analysis of absolute area and volume measurements revealed statistically significant differences among the various groups (p\u0026thinsp;=\u0026thinsp;0.0262 for area and p\u0026thinsp;=\u0026thinsp;0.0170 for volume). Specifically, COHAS_B_P@O displayed a significantly larger increase in bone regeneration area (p\u0026thinsp;=\u0026thinsp;0.0076) and volume (p\u0026thinsp;=\u0026thinsp;0.0019). In contrast, COHAS_B_O had smaller increases in both area and volume. Considering the delayed release kinetics of OPG-Fc, which saturates by week 8 (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec), these result support the hypothesis that sequential release of BMP-2 and OPG-Fc from COHAS enhances bone healing at the defect site. Other groups, except for the volume in COHAS_P@B_O (p\u0026thinsp;=\u0026thinsp;0.0483), did not show statistical significance (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05). Temporal analysis further demonstrated that COHAS_B_P@O had a significant increase in bone area and volume at both weeks 4 and 8 compared to week 2 (all p-values\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eHistological examination of the calvarial defect site using H\u0026amp;E and MT stains is presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea. The MT staining method highlights non-mineralized osteoid connective tissue in blue, while mineralized bone appears red. Among the experimental groups (COHAS, COHAS_B, COHAS_O, COHAS_B_O, COHAS_P@B_O), pronounced infiltration of inflammatory cells was observed in the central defect region. In contrast, the COHAS_B_P@O group showed minimal inflammatory cell presence. Although the extent of bone maturation varied across groups, all exhibited some degree of maturation resembling normal bone structure. Notably, the COHAS_B_P@O group displayed the highest level of bone maturation, as indicated by the larger red-stained area in the MT staining (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003eb).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eTo date, an effective approach to treat critical bone defects remains elusive. Autologous bone transplantation is considered the gold standard for bone regeneration. However, its widespread application is limited by significant time and cost constraints\u003csup\u003e\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e. Moreover, fractures in the elderly are expected to increase due to the global aging population. This trend is accompanied by a rise in age-related bone diseases such as osteopenia, osteoarthritis, and osteoporosis\u003csup\u003e\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u003c/sup\u003e. Hence, there is a pressing need for alternative, more practical solutions for bone healing.\u003c/p\u003e\u003cp\u003eDuring post-fracture bone healing, the inflammatory response remains active for up to five days. This phase is followed by osteoblast activity, which lasts for about ten days. Afterward, osteoclasts collaborate with osteoblasts to remove residual material. Maintaining a balanced ratio between osteoclasts to osteoblasts is crucial for bone health. In patients with osteoporosis, however, the proportion of osteoclasts is elevated relative to osteoblasts. Because of this imbalance, inhibiting osteoclasts is a well-established strategy to prevent osteoporosis. Many studies suggest that bone-replaceable scaffolds, designed to mimic the natural bone healing process, can enhance new bone formation and mitigate associated side effects\u003csup\u003e\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e. In these studies, scaffolds loaded with bioactive molecules have been developed to support bone healing by releasing essential components. However, simultaneous release of these factors may not provide sufficient bone healing efficacy and could even be harmful. Bone regeneration requires about two weeks for optimal osteoblast activation. Therefore, BMP-2 needs both an initial burst release and sustained delivery. Previous research has mainly focused on temporal release, but bulk drug administration has sometimes caused ectopic bone formation\u003csup\u003e\u003cspan additionalcitationids=\"CR49\" citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e. Additionally, administering OPG-Fc alone has shown limited success in promoting bone generation\u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e. To address these issues, we developed scaffolds that sequentially release BMP-2 and OPG-Fc. This approach aims to synergistically improve bone regeneration.\u003c/p\u003e\u003cp\u003eThe key component enabling the delayed release of bioactive molecules is PLGA. It is widely used as a drug carrier due to its biodegradable and biocompatible properties\u003csup\u003e\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u003c/sup\u003e. We confirmed that BMP-2 and OPG-Fc were successfully incorporated into PLGA microspheres, with encapsulation rates of 86.9% and 81.9%, respectively. In Figs.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ec and \u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003ed, we compared the total released amounts of BMP-2 in COHAS_B and COHAS_P@B, as well as OPG-Fc in COHAS_O and COHAS_P@O. This analysis verified that the COHAS components, collagen and hydroxyapatite, do not interfere with PLGA or impede the release of BMP-2 and OPG-Fc. Among the experimental groups shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e, COHAS_B_P@O achieved the most notable result, demonstrating exceptional new bone formation through the delayed release of OPG-Fc. The release profile of OPG-Fc was more delayed compared to that of BMP-2. This difference arises because OPG-Fc is encapsulated within both PLGA microspheres and COHAS, whereas BMP-2 is embedded only in COHAS. Especially, the absolute area and volume of new bone in COHAS_B_P@O exceeded those in COHAS_P@B_O. This result highlights the importance of the drug release sequence in the bone healing process. It also aligns with previous studies regarding the biological stages of fracture healing\u003csup\u003e\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. Specifically, the initial release of BMP-2 stimulates the preosteoblasts. The delayed release of OPG-Fc then inhibits osteoclastogenesis of presoteoclasts in the bone defect microenvironment. Figure S3 shows that only BMP-2 increases ALP activity in MC3T3-E1 cells, while OPG-Fc does not. This is consistent with the results presented in Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e. Furthermore, COHAS_B exhibited significantly greater absolute bone area and volume compared to COHAS_P@B_O. This highlights the importance of properly configuring the release sequence. An improper sequence of BMP-2 and OPG-Fc may negatively affect bone regeneration, potentially more than releasing BMP-2 alone.\u003c/p\u003e\u003cp\u003eOur study focuses on the delivery of multiple drugs and holds significant potential for broader applications in the therapeutic applications, such as wound healing. However, there are limitations to the use of COHAS. Firstly, the bone defect model we used targets cranial bone defects. These defects have minimal load-bearing requirements and lack motion compared to joints or other mobile body parts. In future studies, we plan to enhance the applicability of COHAS by combining it with supportive structures, such as metal frameworks, to provide mechanical strength for load-bearing sites like the knee and spine. Second, the optimal drug loading amount should be customized to suit different bone defect cases, considering variations in size, depth, and severity. Accordingly, we will perform studies in large animal models, including rabbits and pigs, to optimize drug loading according to body weight or defect size. These studies will include long-term evaluations to assess complete bone regeneration. Additionally, the choice or combination of drugs in COHAS may need adjustment depending on the specific bone defect for optimal efficacy.\u003c/p\u003e"},{"header":"5. Conclusion","content":"\u003cp\u003eWe fabricated COHAS and developed a sequential drug release system that co-loads osteoblast- stimulating BMP-2 and osteoclast-inhibiting OPG-Fc. We confirmed that BMP-2 enhances osteogenic activity in osteoblasts, while OPG-Fc effectively inhibits preosteoclast differentiation in vitro. Inspired by the natural bone healing process, in which bone resorption is orchestrated by prolonged osteoclast differentiation and activation, we encapsulated OPG-Fc within PLGA microspheres for sustained and delayed release. This approach produced a synergistic bone healing effect when combined with BMP-2. Our results demonstrate that COHAS_B_P@O effectively promotes bone healing by activating osteoblasts throughout bone regeneration and inhibiting osteoclast precursor differentiation from the intermediate stage. Also, all COHAS formulations co-loaded with drugs showed no cytotoxicity and exhibited good biocompatibility, which are essential for biomaterial utilization. We believe that our dual-drug delivery platform of BMP-2 and OPG-Fc can contribute to harnessing the potential of sequential drug release scaffolds for bone regeneration.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eDeclaration of competing interest\u003c/h2\u003e\u003cp\u003eThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u003c/p\u003e\u003ch2\u003eFunding\u003c/h2\u003e\u003cp\u003eThis work was supported by the National Research Foundation of Korea (NRF) grants from the Korean Government (MSIP) (Grant Nos. 2022R1A6A3A13072828, 2022R1A2C1091756, RS-2024-00400563, RS-2024-00414209, 2018M3C1B7020716, 2018M3C1B7020722, 2019M3C1B7026601). Amgen Inc provided the OPG-Fc under a research program agreement (No. 10655700). This work was supported by BK21 FOUR Institute of Precision Public Health. This work was also supported from Hyundai Motor Chung Mong-Koo Foundation.\u003c/p\u003e\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eAuthor ContributionsJ.W.J., H.K., and J.S.O. contributed equally to this work. H.S., C.N.S., S.H.K., H.K., and D.S.Y. performed funding acquisition, project administration, supervision. J.W.J and H.K. fabricated the drug-loaded scaffolds. J.W.J., H.K., D.L., H.G.J., Y.H.K. and J.S.Y. characterized the drug-loaded scaffolds and analyzed the data. H.J.C. and C.N.S. provided the cell lines. J.S.O., and X.J. performed the animal experiments and analyzed the data. J.W.J., H.K., and J.S.O. co-wrote the original draft. D.L. and D.S.Y. reviewed and edited the draft.\u003c/p\u003e\u003ch2\u003eData Availability\u003c/h2\u003e\u003cp\u003eThe data that support the findings of this study are available within the article and its supplementary information files, and are also available from the corresponding author upon reasonable request.\u003c/p\u003e\n\u003ch2\u003eAUTHOR INFORMATION\u003c/h2\u003e\n\u003cp\u003e\u003cstrong\u003eCorresponding Author\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDae Sung Yoon - School of Biomedical Engineering, Korea University, Seoul 02841, South Korea; \u0026nbsp;Interdisciplinary Program in Precision Public Health, Korea University, Seoul 02841, South Korea; \u0026nbsp;ASTRION, 47, Gaeunsa-gil, Seongbuk-gu, Seoul 02842, Republic of Korea; \u0026nbsp;Email: [email protected]\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eBouvard, B., Annweiler, C. \u0026amp; Legrand, E. 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PLGA based drug carrier and pharmaceutical applications: the most recent advances. \u003cem\u003ePharmaceutics\u003c/em\u003e \u003cb\u003e12\u003c/b\u003e, 903 (2020).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"bone regeneration, sequential drug delivery, bioinspired scaffold","lastPublishedDoi":"10.21203/rs.3.rs-7438279/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7438279/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThe global aging population has raised concerns regarding age-related health issues like osteoporosis and bone fractures. To address these conditions, bone-like scaffolds containing bioactive molecules and biomaterials have been widely studied. However, uncontrolled burst release and delivery of drugs can incur negative side effects. To overcome this issue, a collagen-hydroxyapatite scaffold (COHAS) that can sequentially deliver Bone morphogenetic protein-2 (BMP-2) and Osteoprotegerin fused to the Fc region of immunoglobulin (OPG-Fc) is synthesized. The COHAS comprises a collagen-hydroxyapatite matrix containing BMP-2 and numerous poly-lactic glycolic acid (PLGA) microspheres with OPG-Fc, dispersed in the matrix. The dispersion of PLGA microspheres enables the retardation of OPG-Fc release compared to BMP-2 release. The controlled sequential delivery of BMP-2 and OPG-Fc exhibits synergistic potential in promoting new bone formation by simultaneously activating osteoblasts and deactivating osteoclasts. This investigation revealed that the COHAS co-loaded with BMP-2 and OPG-Fc possesses excellent cell viability and enhanced osteogenic properties in vitro. In vivo assessment via implantation of the drug-loaded COHAS using an 8 mm-calvarial defect rat model demonstrated high efficacy of new bone formation with good biocompatibility. Hence, these findings provide valuable insights for developing therapeutic scaffolds capable of sequential release of multiple drugs, with the potential to extend a cell-free treatment system for bone regeneration.\u003c/p\u003e","manuscriptTitle":"Synergistic Bone Regeneration Through Sequential Dual-drug Delivery","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-18 06:53:11","doi":"10.21203/rs.3.rs-7438279/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"66a41867-bdb4-4068-a0f9-58a2b35fe56c","owner":[],"postedDate":"September 18th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":54815038,"name":"Biological sciences/Biotechnology"},{"id":54815039,"name":"Biological sciences/Drug discovery"},{"id":54815040,"name":"Physical sciences/Materials science"},{"id":54815041,"name":"Health sciences/Medical research"}],"tags":[],"updatedAt":"2025-10-01T05:08:25+00:00","versionOfRecord":[],"versionCreatedAt":"2025-09-18 06:53:11","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7438279","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7438279","identity":"rs-7438279","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}