Intraperitoneal clodronate liposomes remodel the local macrophage niche and potentiate biomaterial-induced osteoinduction

Intraperitoneal clodronate liposomes remodel the local macrophage niche and potentiate biomaterial-induced osteoinduction | 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 Research Article Intraperitoneal clodronate liposomes remodel the local macrophage niche and potentiate biomaterial-induced osteoinduction Wei Cao, Zhiqiao Hu, Xiaodong Guo, Mingzheng Li, Zhangling Nie, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7688492/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 09 Feb, 2026 Read the published version in Molecular Biology Reports → Version 1 posted 7 You are reading this latest preprint version Abstract Background Osteoinductive biomaterials hold promise for repairing critical-size bone defects, yet the mechanisms underlying materials-instructed osteoinduction (MIOI) remain unclear. Macrophages are central, and local liposomal clodronate (LipClod) typically suppresses osteogenesis. Whether systemic LipClod modulates MIOI is unknown. We investigated the effect of intraperitoneal (i.p.) LipClod on ectopic osteoinduction. Methods Male FVB mice received subcutaneous β-tricalcium phosphate (TCPs) scaffolds and a single i.p. LipClod dose 1 day after implantation; vehicle liposomes served as controls (n = 6 per group; 10 µL/g). Recombinant macrophage colony-stimulating factor (M-CSF; 0.5 or 2 µg/mL; 100 µL per pocket) was injected locally, with contralateral pockets receiving PBS. Outcomes were assessed by histology, immunohistochemistry, RT-qPCR, flow cytometry, and Transwell assays of macrophage-conditioned media on bone marrow mesenchymal stem cell (BMSC) migration and osteogenic genes. Results i.p. LipClod enhanced ectopic bone formation at 8 weeks, supported by histology and increased bone sialoprotein transcripts. Rather than depleting macrophages, it expanded monocyte–macrophage at the implant site and increased BMSC recruitment. Macrophage-conditioned media promoted BMSC migration in vitro. Local M-CSF further boosted macrophage infiltration, favored reparative polarization, and augmented osteogenesis. Conclusions Systemic LipClod augments MIOI by eliciting compensatory expansion of local macrophages that enhances early BMSC trafficking. Carefully calibrated systemic macrophage-targeted interventions may improve osteoinductive biomaterial performance. Osteogenesis Ectopic Macrophages Mesenchymal Stem Cells Cell Movement Clodronic Acid Immunomodulation Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 1 Introduction Regeneration of critical-sized bone defects remains a major clinical challenge. While autologous bone grafting is the clinical gold standard, it is constrained by donor-site morbidity and limited availability [ 1 , 2 ]. Osteoinductive biomaterials that trigger de novo bone formation at ectopic sites without exogenous cells or growth factors represent a promising alternative [ 3 ]. However, successful clinical translation requires a mechanistic understanding of materials-instructed osteoinduction (MIOI). Increasing evidence indicates that macrophages are early, central regulators of MIOI [ 4 ]. After implantation, circulating monocytes rapidly accumulate on biomaterial surfaces, differentiate into macrophages, and polarize into distinct functional states (e.g., pro-inflammatory M1 and pro-reparative M2) in response to local cues [ 5 – 7 ]. For instance, our previous work demonstrated that scaffold surface topography can direct macrophage polarization to promote or impede osteogenesis [ 8 ]. liposomal clodronate (LipClod) is a widely used pharmacological agent that selectively depletes phagocytic macrophages, enabling causal interrogation of their roles across diverse biological processes. The magnitude and distribution of depletion depend strongly on the route of administration [ 9 – 11 ]. Intravenous delivery produces systemic macrophage depletion, particularly in the liver and spleen, whereas subcutaneous injection confines effects locally [ 12 , 13 ]. This route-dependent activity positions LipClod as a tool to modulate defined macrophage niches and dissect their contributions to tissue regeneration. In bone regeneration studies, local LipClod injection at injury or implant sites is commonly used and has been shown to consistently suppress osteogenesis by eliminating essential resident macrophages [ 14 , 15 ]. By contrast, the consequences of systemic intraperitoneal (i.p.) administration remain unclear and even appear paradoxical. Sandberg et al. reported that i.p. LipClod transiently impaired bone repair, whereas Cho et al. observed increased bone mass in an orthotopic setting using the same regimen [ 16 , 17 ]. These conflicting findings, derived exclusively from orthotopic models, underscore a critical knowledge gap: the impact of systemic macrophage modulation via i.p. LipClod on the distinct process of MIOI remains unknown. In this study, one kind of classical osteoinductive β-tricalcium phosphate (TCP) ceramic named TCPs was employed. Our previous studies have proved that TCPs could result in favorable bone formation at nonosseous site[ 8 , 18 , 19 ]. We initially hypothesized that systemic macrophage depletion by i.p. LipClod would suppress MIOI. Unexpectedly, a single post-implantation i.p. dose markedly enhanced ectopic bone formation. This finding prompted mechanistic analyses of local cellular and molecular responses to identify the basis of the pro-osteogenic effect. Here, we reveal a route-specific immunomodulatory mechanism whereby systemic perturbation of the macrophage compartment can be exploited to improve the performance of osteoinductive biomaterials. 2 Materials and methods 2.1 Materials and reagents Porous TCPs (4 × 4 × 4 mm) with submicron-scale surface topography (Fig. 1 a) were supplied by Kuros Biosciences B.V. (Bilthoven, the Netherlands). Scaffolds were sterilized by autoclaving at 121°C for 30 min before use. LipClod and vehicle liposomes containing phosphate-buffered saline (LipPBS) were obtained from Vrije Universiteit Amsterdam (Amsterdam, the Netherlands). All other reagents were of analytical grade or higher. 2.2 In vivo studies 2.2.1 Animals Male FVB mice (5–6 weeks old) were purchased from Charles River Laboratories (Beijing, China). Mice were maintained under specific pathogen-free (SPF) conditions with ad libitum access to food and water. 2.2.2 Surgical procedures All animal procedures complied with the ARRIVE guidelines and were approved by the Animal Care and Use Committee of Sichuan University. A dorsal subcutaneous implantation model was used as previously described [ 8 , 18 , 19 ]. Briefly, anesthesia was induced and maintained with 2% isoflurane, and buprenorphine (0.1 mg/kg) was administered subcutaneously (s.c.) for analgesia. After aseptic preparation of the dorsal skin, 2–4 subcutaneous pockets were created, and one TCPs was inserted into each pocket. 2.2.3 Systemic LipClod and vehicle administration 1 day after implantation, mice received a single i.p. injection of LipClod at 10 µL/g body weight. Control animals received an equal volume of LipPBS. For biodistribution analysis, a separate cohort received a single i.p. injection of fluorescently labeled LipPBS (10 µL/g). 2.2.4 Local M-CSF administration To augment local macrophage numbers, we performed a validation experiment with M-CSF (PeproTech, USA) as previously described [ 20 ]. Scaffolds were injected locally with M-CSF (100 µL; 0.5 or 2.0 µg/mL), whereas contralateral pockets received an equal volume of PBS as controls. 2.2.5 Scaffold harvesting and processing At predefined time points, scaffolds were explanted for analysis. For flow cytometry, explants were dissected free of surrounding tissue and enzymatically digested in RPMI containing 0.5 mg/mL Liberase TL (Sigma, Germany) at 37°C for 45 min with agitation. Cell suspensions were centrifuged (1000 rpm, 5 min), resuspended in PBS, and filtered through a 100 µm strainer to obtain single-cell suspensions. For other analyses, explants were fixed in 4% formaldehyde for histology or snap-frozen and stored at − 80°C for RNA isolation. 2.3 In vitro studies 2.3.1 Isolation and culture of bone marrow-derived macrophages (BMMs) Detailed methods are described in our recent work [ 4 ]. In brief, complete growth medium (GM) consisting of Minimum Essential Medium α (α-MEM; Gibco, USA), 10% fetal bovine serum (FBS; Gibco, USA), and 1% (v/v) penicillin/streptomycin, supplemented with 100 ng/mL M-CSF (AF-315-02-100; PeproTech, USA), was prepared. Cells harvested from the bone marrow were then washed with PBS and cultured in GM in an incubator at 37°C with 5% CO2. After 24 h, the cell suspension was transferred to new culture flasks and cultured for an additional 2 days to allow BMMs to adhere. Finally, a single-cell BMM suspension was prepared by gently scraping. 2.3.2 Murine bone marrow mesenchymal stem cell (mBMSC) migration assay BMMs at different densities (2.5 × 10 5, 5 × 10 5 , and 1.0 × 10 6 cells per disc) were seeded on TCPs. Then, 50 ng/mL M-CSF was added to the medium, and the cells and TCPs scaffolds were co-cultured for 4 days. The supernatant was collected and mixed 1:1 with GM to generate conditioned medium (CM). A 24-well Transwell system (8 µm pore size; Corning, USA) was used to assess mBMSC migration. Briefly, mBMSCs (CRL-12424) at 2 × 10 4 cells per well in serum-free DMEM were seeded into the upper chamber, and 500 µL of CM was added to the lower chamber. After 16 h, cells that migrated to the lower chamber were stained with DAPI, imaged using an inverted fluorescence microscope, and quantified in ImageJ (n = 3). 2.3.3 mBMSC osteogenic differentiation assay To assess osteogenic differentiation, mBMSCs (2 × 10 4 ) were cultured for 7 days in CM prepared as above; cells maintained in standard complete medium served as controls. After 7 days, cells were harvested for RT-qPCR (n = 4). 2.4 Evaluation 2.4.1 Histological analysis Fixed samples were processed for either undecalcified or decalcified sectioning. For undecalcified sections, samples were dehydrated through graded ethanol, embedded in polymethyl methacrylate, sectioned at 10–20 µm, and stained with methylene blue and basic fuchsin. For decalcified sections, samples were treated in 0.5 M EDTA for 10–14 days, dehydrated, and embedded in paraffin; 4–7 µm sections were cut and stained with Masson’s trichrome. All sections were imaged with a light microscope (Leica, Germany). 2.4.2 In vivo fluorescence imaging Mice receiving fluorescently labeled liposomes were anesthetized with 2% isoflurane and imaged at 1, 2, 3, and 4 days post-injection in supine and prone positions using an IVIS Lumina III system (PerkinElmer, USA) 2.4.3 Immunohistochemistry (IHC) Deparaffinized sections underwent antigen retrieval in citrate buffer (95°C, 5 min). Endogenous peroxidase activity was quenched and non-specific binding was blocked with goat serum. Sections were incubated overnight at 4°C with primary antibodies against F4/80 (1:100; Abcam, ab100790), CCR7 (1:50; Novus Biologicals, NB100-712), CD163 (1:200; Abcam, ab182422), or CD206 (HuaBio, JF0953). After incubation with appropriate secondary antibodies, signals were developed using a DAB chromogen kit and counterstained with hematoxylin. 2.4.4 Flow cytometry Single-cell suspensions were permeabilized using a Foxp3/Transcription Factor Staining Buffer Set (eBioscience) and incubated with fluorochrome-conjugated monoclonal antibodies at 4°C for 40 min. Mesenchymal stem cells (MSCs) were gated as CD29 + /CD44 + /CD90 + and CD34 − /CD45 − . Data were acquired on a ZE5 Cell Analyzer (Bio-Rad) and analyzed in Kaluza (n = 4) [ 21 ]. 2.4.5 RT-qPCR Samples were thoroughly pulverized in a glass homogenizer at 4°C. Total RNA was isolated using TRIzol reagent (Thermo Fisher Scientific, USA) according to the manufacturer’s instructions. RNA concentration was measured on a NanoDrop spectrophotometer (Thermo Fisher Scientific, USA). cDNA was synthesized using the RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, USA) following the manufacturer’s instructions. qPCR was performed on an ABI StepOnePlus™ Real-Time PCR System with an initial denaturation at 95°C for 10 min, followed by 40 cycles of 95°C for 30 s, 60°C for 1 min, and 72°C for 1 min. GAPDH was chosen as the housekeeping gene for normalization. BSP was initially assessed to evaluate variations in TCPs-instructed osteogenesis under the influence of LipClod or LipPBS. For macrophage polarization, expression of F4/80 (a pan-macrophage marker), CCR7/iNOS (M1 markers), and Arg1/CD163/CD206 (M2 markers) was evaluated; BSP was also quantified. For mBMSC osteogenic differentiation, gene expression of ALP, OCN, OPN, Runx2, Osterix, and Col1a1 was investigated. For each gene, 4–5 samples were analyzed, and each sample was assayed in triplicate. Relative gene expression was calculated using the 2^−ΔΔCt method. 2.5 Statistical analysis Data are presented as mean ± standard deviation (SD). Statistical significance was determined using Student’s t test for two-group comparisons or one-way analysis of variance (ANOVA) with Tukey’s post hoc test for multiple comparisons. A p value < 0.05 was considered statistically significant (p < 0.05; *p < 0.01; **p < 0.001; ***p < 0.0001). 3 Results 3.1 i.p. LipClod administration enhances TCPs-instructed ectopic osteogenesis Eight weeks post-implantation, significant osteogenesis was observed in the LipPBS control group (Fig. 1b1, 1c1, 1c1′), confirming the favorable osteoinductive properties of the TCPs scaffold. Compared with LipPBS, LipClod treatment significantly promoted new bone formation (Fig. 1b2, 1c2, 1c2′, 1d), indicating that i.p. LipClod enhances TCPs-instructed osteoinduction. Furthermore, RT-qPCR showed a significant upregulation of bone sialoprotein (BSP) transcripts following i.p. LipClod administration, a molecule implicated in the early-stage formation of apatite crystals [ 22 , 23 ]. 3.2 Biodistribution of liposomes following i.p. injection As revealed by fluorescence imaging (Fig. 2 ), following i.p. injection, most liposomes remained localized at the injection site throughout the observation period, with only a small fraction reaching the distal implantation site via the circulation. The fluorescence intensity at the implantation site peaked at day 3 post-injection and subsequently declined, suggesting clearance of liposomes, potentially via the kidneys. 3.3 i.p. LipClod injection modulates macrophage recruitment and polarization At day 3 post-injection, immunostaining (Fig. 3 ) revealed more CD11b + , F4/80 + , and CCR7 + cells in the LipClod group than in LipPBS, whereas CD206 + (M2) cells did not differ between groups. At the transcript level (Fig. 4 a), LipClod increased F4/80 and iNOS (M1) expression. Despite comparable CD206 + counts by IHC, Arg1 and CD206 transcripts were also elevated in the LipClod group [ 24 – 26 ]. 3.4 i.p. LipClod administration promotes mBMSC migration To assess whether i.p. LipClod affects mBMSC migration, we quantified migrated cells using flow cytometry in Transwell assays. Compared with LipPBS controls (20.28%), i.p. LipClod increased the proportion of migrated mBMSCs to 29.48% (Fig. 4 b, 4 c). 3.5 Local M-CSF injection augments osteogenesis and modulates macrophage behavior Histology showed that local M-CSF enhanced ectopic osteogenesis in a concentration-dependent manner (Fig. 5 a); the higher dose (2 µg/mL) produced significantly more new bone than control (Fig. 5 b). Concordantly, IHC and RT-qPCR demonstrated dose-dependent recruitment of F4/80 + macrophages (Fig. 6 ). Notably, increased recruitment coincided with fewer CCR7 + M1 macrophages, suggesting that M-CSF also modulates polarization [ 29 ]. RT-qPCR further showed a negative correlation between CCR7 transcripts and M-CSF dose, whereas CD206 (M2) expression increased with M-CSF concentration. 3.6 Effect of macrophage population on the migration and osteogenic differentiation of mBMSCs The Transwell migration assay demonstrated that CM from co-cultures with higher macrophage populations significantly promoted migration of murine bone marrow–derived mBMSCs (Fig. 7 a). In contrast, for osteogenic differentiation, CM from any macrophage density did not significantly alter expression of ALP, OCN, or Osterix compared with controls (Fig. 7 b). Furthermore, CM treatment significantly decreased expression of OPN, Col1a1, and Runx2 relative to controls. Among the different macrophage-density groups, a significant difference was observed only for Runx2 between the 2.5 × 10⁵ and 1.0 × 10⁶ cells per disc groups. 4 Discussion Conventionally, macrophage depletion using LipClod is considered detrimental to MIOI [ 8 , 15 ]. In contrast, we found that i.p. LipClod promoted TCPs-instructed ectopic osteogenesis, indicating a route-dependent host response. Liposome biodistribution tracking offers a mechanistic explanation. After i.p. injection, most liposomes (LipClod) remained within the peritoneal cavity and only a minor fraction reached distal implants, consistent with liver and spleen accumulation and low plasma levels reported by Ylitalo [ 27 ]. Consequently, drug levels at the implant are too low and transient for durable macrophage depletion, potentially modulating rather than ablating local macrophages. At the implant site, we observed increased CD11b + and F4/80 + cells with broad upregulation of M1 (CCR7, iNOS) and M2 (CD163, Arg1, CD206) markers, indicating robust monocyte recruitment and differentiation and a mixed pro-inflammatory/reparative milieu. These changes align with a systemic compensation model. i.p. LipClod depletes macrophages in peritoneal and omental niches within days [ 28 , 29 ], prompting increased bone marrow monocyte output and peripheral mobilization; newly generated cells home to injured scaffolds in response to inflammatory cues [ 30 – 32 ]. Consistently, long-term i.p. LipClod treatment increased tibial trabecular bone mass and potentiated intermittent parathyroid hormone responses with enrichment of CD68 + /CD163 + myeloid cells and upregulation of osteogenic genes (Wnt3a, Wnt10b, Tgfb1) [ 17 ]. In a tooth-extraction model, site-dependent effects were observed: tibial BV/TV rose by 81% (day 7) and 152% (day 14) versus PBS, whereas oral socket bone fill increased by ~ 7% (day 14), with steady-state oral bone unchanged [ 33 ]. Moreover, i.p. LipClod directly affects bone marrow monocytes, decreasing mature and increasing immature subsets for ~ 3–5 days [ 16 ]. Thus, any limited local depletion is rapidly offset by influx and local proliferation of newly recruited macrophages. An expanded macrophage population appears to promote osteogenesis primarily by enhancing MSC recruitment [ 34 ]. CM from pro-inflammatory or repair-associated macrophages stimulates MSC recruitment, with stronger effects for pro-inflammatory CM—likely via factors such as OSM, TNF-α, and IL-10 that drive MSC elongation [ 35 ]; M1-like macrophages are particularly important for this process [ 36 ]. In our in vitro experiments, CM from high-density macrophage cultures significantly promoted MSC migration, whereas it either failed to increase or even decreased expression of osteogenic markers. This observation is consistent with previous evidence showing that pro-inflammatory macrophage CM favors MSC migration, whereas anti-inflammatory factors are more closely associated with osteogenic differentiation [ 35 ]. Moreover, mechanistic studies have demonstrated that macrophage-derived chemokines such as CCL5, CCL2, and IL-8 can drive a ~ 5–9-fold increase in MSC chemotaxis through JNK signaling [ 38 ]. Taken together, these findings and our own results support a model in which i.p. LipClod generates an early, chemokine-rich microenvironment that efficiently recruits host MSCs, thereby establishing the cellular basis for subsequent osteogenesis. To directly test the “numbers matter” hypothesis, local injections of M-CSF increased macrophage infiltration and enhanced bone formation in a dose-dependent manner [ 39 ]. M-CSF also biased polarization toward an M2 phenotype and underscored that macrophage abundance and timing, rather than any single polarization state, govern the pro-osteogenic niche [40]. Together, these findings support a route-dependent model of clearance followed by compensation: i.p. delivery confines most liposomes to the peritoneal compartment, limiting distal depletion; transient loss of peritoneal and omental macrophages drives systemic myelopoiesis and monocyte recruitment to implants; the ensuing early, mixed M1/M2, chemokine-rich milieu primarily enhances MSC homing with limited direct effects on osteogenic differentiation. This framework predicts that attenuating early inflammatory cues (24–72 h) will blunt MSC homing and weaken MIOI, whereas interventions at later stages will have smaller effects; it also highlights dose and route of administration as actionable levers to engineer osteoinductive immunity. 5 Conclusion This study demonstrates that i.p. delivery of LipClod paradoxically enhances TCPs-instructed ectopic osteogenesis by increasing peri-implant macrophage abundance. We propose that this systemic stimulus elicits a compensatory host response that accelerates production and preferential homing of monocyte–macrophage precursors to the implant site (Scheme 1 ). This, in turn, enhances recruitment of MSCs—crucial for osteogenesis—likely via pro-inflammatory cytokines. Collectively, our findings present a route-specific strategy to potentiate MIOI by modulating systemic host responses and provide evidence that macrophage abundance is a key determinant in osteoimmunology. These insights rationalize the development of immunomodulatory biomaterial strategies to enhance bone regeneration and may help accelerate clinical translation. Declarations Funding: This research was supported by grants from the National Natural Science Foundation of China (Grant No. 82371013) and the Research and Development Program of the West China Hospital of Stomatology, Sichuan University (Grant No. RD-03-202306). Competing interests: The authors declare no competing interests. Ethics approval: All animal procedures were approved by the Animal Care and Use Committee of Sichuan University (Approval No. WCHSIRB-D-2024-525). Consent to participate: Not applicable. Consent for publication: Not applicable. Data availability : All data supporting the findings of this study are included in the article. Raw datasets are available from the corresponding author upon reasonable request. Authors’ contributions: Wei Cao and Zhiqiao Hu performed the investigation and drafted the original manuscript. Xiaodong Guo contributed to methodology and investigation. Mingzheng Li, Zhangling Nie, and Chengen Li performed investigation. Wenting Qi and Yue Wang performed histological assessments. Yu Xiao led conceptualization, provided supervision, and handled writing—review and editing. Chongyun Bao secured funding (funding acquisition). All authors commented on previous versions of the manuscript, and all authors read and approved the final manuscript. References Roddy E, DeBaun MR, Daoud-Gray A, Yang YP, Gardner MJ (2018) Treatment of critical-sized bone defects: clinical and tissue engineering perspectives. Eur J Orthop Surg Traumatol 28:351–362. https://doi.org/10.1007/s00590-017-2063-0 Schmidt AH (2021) Autologous bone graft: is it still the gold standard? Injury 52(Suppl 2):18–22. https://doi.org/10.1016/j.injury.2021.01.043 Bai H, Guo X, Tan Y, Wang Y, Feng J, Lei K, Liu X, Xiao Y, Bao C (2022) Hypoxia-inducible factor-1 signaling pathway in macrophage-involved angiogenesis in materials-instructed osteoinduction. J Mater Chem B 10:6483–6495. https://doi.org/10.1039/d2tb00811d Bohner M, Miron RJ (2019) A proposed mechanism for material-induced heterotopic ossification. Mater Today 22:132–141. https://doi.org/10.1016/j.mattod.2018.10.036 Pajarinen J, Lin T, Gibon E, Kohno Y, Maruyama M, Nathan K, Lu L, Yao Z, Goodman SB (2019) Mesenchymal stem cell–macrophage crosstalk and bone healing. Biomaterials 196:80–89. https://doi.org/10.1016/j.biomaterials.2017.12.025 Lee J, Byun H, Madhurakkat Perikamana SK, Lee S, Shin H (2019) Current advances in immunomodulatory biomaterials for bone regeneration. Adv Healthc Mater 8:e1801106. https://doi.org/10.1002/adhm.201801106 Newman H, Shih YV, Varghese S (2021) Resolution of inflammation in bone regeneration: from understandings to therapeutic applications. Biomaterials 277:121114. https://doi.org/10.1016/j.biomaterials.2021.121114 Li M, Guo X, Qi W, Wu Z, de Bruijn JD, Xiao Y, Bao C, Yuan H (2020) Macrophage polarization plays roles in bone formation instructed by calcium phosphate ceramics. J Mater Chem B 8:1863–1877. https://doi.org/10.1039/c9tb02932j Alexander KA, Chang MK, Maylin ER, Kohler T, Muller R, Wu AC, van Rooijen N, Sweet MJ, Hume DA, Raggatt LJ, Pettit AR (2011) Osteal macrophages promote in vivo intramembranous bone healing in a mouse tibial injury model. J Bone Miner Res 26:1517–1532. https://doi.org/10.1002/jbmr.354 van Rooijen N, Hendrikx E (2010) Liposomes for specific depletion of macrophages from organs and tissues. Methods Mol Biol 605:189–203. https://doi.org/10.1007/978-1-60327-360-2_13 McCloskey E, Paterson AH, Powles T, Kanis JA (2020) Clodronate. Bone 143:115715. https://doi.org/10.1016/j.bone.2020.115715 Michalski MN, Zweifler LE, Sinder BP, Koh AJ, Yamashita J, Roca H, McCauley LK (2019) Clodronate-loaded liposome treatment has site-specific skeletal effects. J Dent Res 98:459–467. https://doi.org/10.1177/0022034518821685 Okada E, Nakata H, Yamamoto M, Kasugai S, Kuroda S (2018) Indirect osteoblast differentiation by liposomal clodronate. J Cell Mol Med 22:1127–1137. https://doi.org/10.1111/jcmm.13366 Dai B, Xu J, Li X, Huang L, Hopkins C, Wang H, Yao H, Mi J, Zheng L, Wang J, Tong W, Chow DH-K, Li Y, He X, Hu P, Chen Z, Zu H, Li Y, Yao Y, Jiang Q, Qin L (2022) Macrophages in epididymal adipose tissue secrete osteopontin to regulate bone homeostasis. Nat Commun 13:427. https://doi.org/10.1038/s41467-021-27683-w Davison NL, Gamblin AL, Layrolle P, Yuan H, de Bruijn JD, Barrere-de Groot F (2014) Liposomal clodronate inhibition of osteoclastogenesis and osteoinduction by submicrostructured beta-tricalcium phosphate. Biomaterials 35:5088–5097. https://doi.org/10.1016/j.biomaterials.2014.03.013 Sandberg OH, Tatting L, Bernhardsson ME, Aspenberg P (2017) Temporal role of macrophages in cancellous bone healing. Bone 101:129–133. https://doi.org/10.1016/j.bone.2017.04.004 Cho SW, Soki FN, Koh AJ, Eber MR, Entezami P, Park SI, van Rooijen N, McCauley LK (2014) Osteal macrophages support physiologic skeletal remodeling and anabolic actions of parathyroid hormone in bone. Proc Natl Acad Sci U S A 111:1545–1550. https://doi.org/10.1073/pnas.1315153111 Guo X, Li M, Qi W, Bai H, Nie Z, Hu Z, Xiao Y, de Bruijn JD, Bao C, Yuan H (2021) Serial cellular events in bone formation initiated by calcium phosphate ceramics. Acta Biomater 134:730–743. https://doi.org/10.1016/j.actbio.2021.07.037 Nie Z, Hu Z, Guo X, Xiao Y, Liu X, de Bruijn JD, Bao C, Yuan H (2023) Genesis of osteoclasts on calcium phosphate ceramics and their role in material-induced bone formation. Acta Biomater 157:625–638. https://doi.org/10.1016/j.actbio.2022.11.005 O’Brien EM, Risser GE, Spiller KL (2019) Sequential drug delivery to modulate macrophage behavior and enhance implant integration. Adv Drug Deliv Rev 149–150:85–94. https://doi.org/10.1016/j.addr.2019.05.005 Wexler SA, Donaldson C, Kendall PD, Rice C, Bradley B, Hows JM (2003) Adult bone marrow is a rich source of human mesenchymal ‘stem’ cells but umbilical cord and mobilized adult blood are not. Br J Haematol 121:368–374. https://doi.org/10.1046/j.1365-2141.2003.04284.x Malaval L, Wade-Gueye NM, Boudiffa M, Fei J, Zirngibl R, Chen F, Laroche N, Roux JP, Burt-Pichat B, Duboeuf F, Boivin G, Jurdic P, Lafage-Proust MH, Amedee J, Vico L, Rossant J, Aubin JE (2008) Bone sialoprotein plays a functional role in bone formation and osteoclastogenesis. J Exp Med 205:1145–1153. https://doi.org/10.1084/jem.20071294 Kruger TE, Miller AH, Godwin AK, Wang J (2014) Bone sialoprotein and osteopontin in bone metastasis of osteotropic cancers. Crit Rev Oncol Hematol 89:330–341. https://doi.org/10.1016/j.critrevonc.2013.08.013 Tsukasaki M, Takayanagi H (2019) Osteoimmunology: evolving concepts in bone–immune interactions in health and disease. Nat Rev Immunol 19:626–642. https://doi.org/10.1038/s41577-019-0178-8 Okamoto K, Nakashima T, Shinohara M, Negishi-Koga T, Komatsu N, Terashima A, Sawa S, Nitta T, Takayanagi H (2017) Osteoimmunology: the conceptual framework unifying the immune and skeletal systems. Physiol Rev 97:1295–1349. https://doi.org/10.1152/physrev.00036.2016 Zhao Q, Liu X, Yu C, Xiao Y (2022) Macrophages and bone marrow-derived mesenchymal stem cells work in concert to promote fracture healing: a brief review. DNA Cell Biol 41:276–284. https://doi.org/10.1089/dna.2021.0869 Monkkonen J, Ylitalo P (1990) The tissue distribution of clodronate (dichloromethylene bisphosphonate) in mice: effects of vehicle and route of administration. Eur J Drug Metab Pharmacokinet 15:239–243. https://doi.org/10.1007/BF03190210 Biewenga J, van der Ende MB, Krist LFG, et al. (1995) Macrophage depletion in the rat after i.p. administration of liposome-encapsulated clodronate: depletion kinetics and accelerated repopulation of peritoneal and omental macrophages by administration of Freund’s adjuvant. Cell Tissue Res 280:189–196. https://doi.org/10.1007/BF00304524 Bu L, Gao M, Qu S, Liu D (2013) Intraperitoneal injection of clodronate liposomes eliminates visceral adipose tissue macrophages. AAPS J 15:1001–1011. https://doi.org/10.1208/s12248-013-9501-7 Louwe PA, Badiola Gomez L, Webster H, Perona-Wright G, Bain CC, Forbes SJ, Jenkins SJ (2021) Recruited macrophages that colonize the post-inflammatory peritoneal niche convert into functionally divergent resident cells. Nat Commun 12:1770. https://doi.org/10.1038/s41467-021-21778-0 Kaur S, Sehgal A, Wu AC, et al. (2021) Stable colony-stimulating factor 1 fusion protein treatment increases hematopoietic stem cell pool and enhances their mobilisation in mice. J Hematol Oncol 14:3. https://doi.org/10.1186/s13045-020-00997-w Oza D, Ivich F, Pace J, Yu M, Niedre M, Amiji M (2024) Lipid nanoparticle–encapsulated large peritoneal macrophages migrate to the lungs via the systemic circulation in a model of clodronate-mediated lung-resident macrophage depletion. Theranostics 14(6):2526–2543. https://doi.org/10.7150/thno.91062 Michalski MN, McCauley LK (2019) Clodronate-loaded liposome treatment has site-specific skeletal effects. JBMR Plus 3(2):e10245. https://doi.org/10.1002/jbm4.10245 Song G, Habibovic P, Bao C, Hu J, van Blitterswijk CA, Yuan H, Chen W, Xu HH (2013) The homing of bone marrow MSCs to non-osseous sites for ectopic bone formation induced by osteoinductive calcium phosphate. Biomaterials 34:2167–2176. https://doi.org/10.1016/j.biomaterials.2012.12.010 Vallés G, Bensiamar F, Maestro-Paramio L, García-Rey E, Vilaboa N, Saldaña L (2020) Influence of inflammatory conditions provided by macrophages on osteogenic ability of mesenchymal stem cells. Stem Cell Res Ther 11:57. https://doi.org/10.1186/s13287-020-1578-1Wasnik S, Rundle CH, Baylink DJ, Yazdi MS, Carreon EE, Xu Y, Qin X, Lau KW, Tang X (2018) 1,25-Dihydroxyvitamin D suppresses M1 macrophages and promotes M2 differentiation at bone injury sites. JCI Insight 3:e98773. https://doi.org/10.1172/jci.insight.98773 Anton K, Banerjee D, Glod J (2012) Macrophage-associated mesenchymal stem cells assume an activated, migratory, pro-inflammatory phenotype with increased IL-6 and CXCL10 secretion. PLoS One 7:e35036. https://doi.org/10.1371/journal.pone.0035036 Starlinger J, Sarahrudi K, Kecht M, Koerbler F, Pietschmann P, Aharinejad S (2021) The influence of M-CSF on fracture healing in a mouse model. Sci Rep 11:22326. https://doi.org/10.1038/s41598-021-01673-w Vogel DYS, Glim JE, Stavenuiter AWD, Breur M, Heijnen P, Amor S, Dijkstra CD, Beelen RHJ (2014) Human macrophage polarization in vitro: maturation and activation methods compared. Immunobiology 219:695–703. https://doi.org/10.1016/j.imbio.2014.05.002 Scheme 1 Scheme 1 is available in the Supplementary Files section. Additional Declarations No competing interests reported. Supplementary Files floatimage8.png Scheme 1 Route-dependent model: i.p. LipClod limits distal depletion, triggers systemic compensation, expands peri-implant macrophages, enhances early BMSC homing, and augments TCPs-instructed osteoinduction (MIOI) Cite Share Download PDF Status: Published Journal Publication published 09 Feb, 2026 Read the published version in Molecular Biology Reports → Version 1 posted Editorial decision: Revision requested 20 Nov, 2025 Reviews received at journal 13 Nov, 2025 Reviewers agreed at journal 10 Nov, 2025 Reviewers invited by journal 21 Oct, 2025 Editor assigned by journal 24 Sep, 2025 Submission checks completed at journal 24 Sep, 2025 First submitted to journal 22 Sep, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7688492","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":532861404,"identity":"77258cb2-9d19-45c9-b28d-1d0501d4725f","order_by":0,"name":"Wei Cao","email":"","orcid":"","institution":"National Clinical Research Center for Oral Diseases, West China Hospital of Stomatology, Sichuan University","correspondingAuthor":false,"prefix":"","firstName":"Wei","middleName":"","lastName":"Cao","suffix":""},{"id":532861405,"identity":"53813ce2-3529-428b-a266-5adfd0a91010","order_by":1,"name":"Zhiqiao Hu","email":"","orcid":"","institution":"National Clinical Research Center for Oral Diseases, West China Hospital of Stomatology, Sichuan University","correspondingAuthor":false,"prefix":"","firstName":"Zhiqiao","middleName":"","lastName":"Hu","suffix":""},{"id":532861406,"identity":"5d2fae44-2aac-43a2-b326-42d622d55781","order_by":2,"name":"Xiaodong Guo","email":"","orcid":"","institution":"National Clinical Research Center for Oral Diseases, West China Hospital of Stomatology, Sichuan University","correspondingAuthor":false,"prefix":"","firstName":"Xiaodong","middleName":"","lastName":"Guo","suffix":""},{"id":532861407,"identity":"e25b0ba1-e419-4399-9abe-4801ad96eec1","order_by":3,"name":"Mingzheng Li","email":"","orcid":"","institution":"National Clinical Research Center for Oral Diseases, West China Hospital of Stomatology, Sichuan University","correspondingAuthor":false,"prefix":"","firstName":"Mingzheng","middleName":"","lastName":"Li","suffix":""},{"id":532861408,"identity":"0fb4201c-93cc-47e2-b541-47f9c6870b1c","order_by":4,"name":"Zhangling Nie","email":"","orcid":"","institution":"National Clinical Research Center for Oral Diseases, West China Hospital of Stomatology, Sichuan University","correspondingAuthor":false,"prefix":"","firstName":"Zhangling","middleName":"","lastName":"Nie","suffix":""},{"id":532861409,"identity":"bfc1ce6d-ee5e-406b-aa2f-091636fe06cb","order_by":5,"name":"Yue Wang","email":"","orcid":"","institution":"National Clinical Research Center for Oral Diseases, West China Hospital of Stomatology, Sichuan University","correspondingAuthor":false,"prefix":"","firstName":"Yue","middleName":"","lastName":"Wang","suffix":""},{"id":532861410,"identity":"7540ae94-bc09-46e3-b4f8-9c4f30bf7c25","order_by":6,"name":"Chengen Li","email":"","orcid":"","institution":"National Clinical Research Center for Oral Diseases, West China Hospital of Stomatology, Sichuan University","correspondingAuthor":false,"prefix":"","firstName":"Chengen","middleName":"","lastName":"Li","suffix":""},{"id":532861411,"identity":"c0762f54-e4c7-478b-ad83-75776ca8b468","order_by":7,"name":"Wenting Qi","email":"","orcid":"","institution":"National Clinical Research Center for Oral Diseases, West China Hospital of Stomatology, Sichuan University","correspondingAuthor":false,"prefix":"","firstName":"Wenting","middleName":"","lastName":"Qi","suffix":""},{"id":532861412,"identity":"1d3f605f-facb-4cf4-9840-00b843a3d184","order_by":8,"name":"Yu Xiao","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA80lEQVRIie3QMQrCMBSA4cRAXaJdEwp6hUrBqeBVIkKnIoLgqhCIS90VPIQuzkrBLlXXjkpXB6Eguhnt4pR2FMwPyRDex4MAoNP9Yuhzu2Ozyrf5y7Yc8RgN9qwkyQuZnfh2OWJH6JLWhNcHcZzdngI06gmD2UBBKDccpybcIZzONnQmgEMThqy5gpgItC25BXJ82CAoQHeVMANhBTFQ9S5JCAXx00yScSExEX5vCbsB8YElifyHAkI5HraWR88heN+mwZG0FvGFWypin6L1+TpyG52Ip7fHyG3Wo94uU5F3la8BIg+cFAA58igc0el0ur/uBTtlSWd1FNwUAAAAAElFTkSuQmCC","orcid":"","institution":"National Clinical Research Center for Oral Diseases, West China Hospital of Stomatology, Sichuan University","correspondingAuthor":true,"prefix":"","firstName":"Yu","middleName":"","lastName":"Xiao","suffix":""},{"id":532861413,"identity":"7cea8d55-5118-440d-952f-0f81f4c37eb5","order_by":9,"name":"Chongyun Bao","email":"","orcid":"","institution":"National Clinical Research Center for Oral Diseases, West China Hospital of Stomatology, Sichuan University","correspondingAuthor":false,"prefix":"","firstName":"Chongyun","middleName":"","lastName":"Bao","suffix":""}],"badges":[],"createdAt":"2025-09-23 02:38:12","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7688492/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7688492/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s11033-026-11533-3","type":"published","date":"2026-02-09T15:58:48+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":94986933,"identity":"062c5311-7a19-45dd-8fc9-afd174cc08ae","added_by":"auto","created_at":"2025-11-03 07:00:59","extension":"docx","order_by":0,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":2081527,"visible":true,"origin":"","legend":"","description":"","filename":"MBRManuscriptCaoClodronateOsteoinduction.docx","url":"https://assets-eu.researchsquare.com/files/rs-7688492/v1/d2405a4da505c3ca2ac745f1.docx"},{"id":94986223,"identity":"37ebb080-6ab3-47e5-8160-e2dd20db647c","added_by":"auto","created_at":"2025-11-03 07:00:05","extension":"json","order_by":1,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":10603,"visible":true,"origin":"","legend":"","description":"","filename":"77c41a7dd90446c89a1fe7f4970cb5c3.json","url":"https://assets-eu.researchsquare.com/files/rs-7688492/v1/8c806592f00df49f91ca775f.json"},{"id":94986225,"identity":"2418bf46-8011-44df-ab27-4647fbd4340c","added_by":"auto","created_at":"2025-11-03 07:00:05","extension":"xml","order_by":2,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":108247,"visible":true,"origin":"","legend":"","description":"","filename":"77c41a7dd90446c89a1fe7f4970cb5c31enriched.xml","url":"https://assets-eu.researchsquare.com/files/rs-7688492/v1/abdec38fd28c27e3afda7df9.xml"},{"id":94986291,"identity":"c54d1233-876e-40d6-abc3-34eeff5e6e28","added_by":"auto","created_at":"2025-11-03 07:00:09","extension":"jpeg","order_by":3,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":252877,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7688492/v1/f12db59f9e329758e68a5cb9.jpeg"},{"id":94985711,"identity":"31a064ae-d6f3-4b92-b371-539dd8644c9e","added_by":"auto","created_at":"2025-11-03 06:58:45","extension":"jpeg","order_by":4,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":117332,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7688492/v1/012e2d9a38475da210cffabb.jpeg"},{"id":94986908,"identity":"4403e8bc-0b5d-40f7-8f38-0f551be344eb","added_by":"auto","created_at":"2025-11-03 07:00:57","extension":"jpeg","order_by":5,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":276842,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7688492/v1/aeb43ff13b1f0786f967643a.jpeg"},{"id":94870561,"identity":"9c35177a-a837-4858-9213-b7d8f63d6d77","added_by":"auto","created_at":"2025-10-31 14:51:55","extension":"jpeg","order_by":6,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":159187,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7688492/v1/c8e7e2fe9e186390fa2f46ff.jpeg"},{"id":94870558,"identity":"3cf0eadf-ea76-4e67-b523-8b7cf4d19975","added_by":"auto","created_at":"2025-10-31 14:51:55","extension":"jpeg","order_by":7,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":340616,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7688492/v1/2913d9f65261f5a8db149960.jpeg"},{"id":94870566,"identity":"e9d0ef74-0921-4528-869b-8978ac0db06a","added_by":"auto","created_at":"2025-10-31 14:51:56","extension":"jpeg","order_by":8,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":283927,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage6.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7688492/v1/1d1144c4c6985a6048d0ea56.jpeg"},{"id":94870554,"identity":"0a6334dd-6f07-44cc-9877-5f243060fc24","added_by":"auto","created_at":"2025-10-31 14:51:55","extension":"jpeg","order_by":9,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":165130,"visible":true,"origin":"","legend":"","description":"","filename":"floatimage7.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7688492/v1/ce5d30aa0552fca25538c332.jpeg"},{"id":94870568,"identity":"437f3fd1-eeb8-4b25-bd85-5b5a178627ea","added_by":"auto","created_at":"2025-10-31 14:51:56","extension":"png","order_by":11,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":271777,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-7688492/v1/e11acae870d95f0104354f4c.png"},{"id":94870570,"identity":"ad77df66-bad8-474a-b2d8-c3dbaefeb880","added_by":"auto","created_at":"2025-10-31 14:51:56","extension":"png","order_by":12,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":83404,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-7688492/v1/42a4382bb1dd0bc222b8445b.png"},{"id":94986550,"identity":"af9b32f4-167a-49f8-8cc5-66329e828530","added_by":"auto","created_at":"2025-11-03 07:00:25","extension":"png","order_by":13,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":217724,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-7688492/v1/7900635d3491e154874567e8.png"},{"id":94870555,"identity":"6893b7e5-345c-445f-b65f-18ca7b47a419","added_by":"auto","created_at":"2025-10-31 14:51:55","extension":"png","order_by":14,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":84085,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-7688492/v1/336a8e49b47a952bca602c00.png"},{"id":94870565,"identity":"f08ef966-ccf5-4be8-9dca-dc7b269eda75","added_by":"auto","created_at":"2025-10-31 14:51:56","extension":"png","order_by":15,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":371816,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-7688492/v1/18922d8d86e6d5489f6d6ba9.png"},{"id":94870571,"identity":"f4dbd0ec-ff63-4f4c-a56c-a647d661f5fe","added_by":"auto","created_at":"2025-10-31 14:51:56","extension":"png","order_by":16,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":198673,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-7688492/v1/2b69e2ee05b4fc1c41fae4a0.png"},{"id":94870567,"identity":"5e4eb318-0df7-45de-8bda-4d3ab15c7633","added_by":"auto","created_at":"2025-10-31 14:51:56","extension":"png","order_by":17,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":122288,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-7688492/v1/5236e615c6b24ac8865f0e36.png"},{"id":94870562,"identity":"04168f64-b67b-44e7-a678-9b109683dbe2","added_by":"auto","created_at":"2025-10-31 14:51:55","extension":"png","order_by":18,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":66284,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage8.png","url":"https://assets-eu.researchsquare.com/files/rs-7688492/v1/b182e625e45449ed109e6658.png"},{"id":94870563,"identity":"c00ba69d-f54f-428a-930f-137fec003842","added_by":"auto","created_at":"2025-10-31 14:51:56","extension":"xml","order_by":19,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":108610,"visible":true,"origin":"","legend":"","description":"","filename":"77c41a7dd90446c89a1fe7f4970cb5c31structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-7688492/v1/c357b0e7fd2ed25c566fedbd.xml"},{"id":94870569,"identity":"7457e20c-785d-4e6a-8d3e-ef93b9fe416a","added_by":"auto","created_at":"2025-10-31 14:51:56","extension":"html","order_by":20,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":117111,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7688492/v1/3be648a1d8e5cf40f14cda8b.html"},{"id":94870543,"identity":"8d628d57-20f2-4685-aac0-838fe26817c9","added_by":"auto","created_at":"2025-10-31 14:51:55","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":543942,"visible":true,"origin":"","legend":"\u003cp\u003e(a) Surface topography of TCPs; Scale bar = 5 µm (b) H\u0026amp;E staining and (c) Masson’s trichrome staining of TCPs harvested 8 weeks after implantation in mice receiving i.p. LipPBS (b1, c1) or LipClod (b2, c2); magnified regions are shown in c1′ and c2′ (red dashed boxes). Scale bars: b1 and b2, 200 µm; c1 and c2, 400 µm; c1′ and c2′, 100 µm. Black pentagrams denote scaffold material exhibiting bone induction, and green arrows indicate newly formed bone tissue (d) Percentage of new bone in LipPBS and LipClod groups 8 weeks after implantation (e) RT-qPCR of BSP transcripts in LipPBS and LipClod groups 8 weeks after implantation\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-7688492/v1/5f856346ec4a6aae47329364.png"},{"id":94870548,"identity":"ff928806-75b9-458e-bbab-3db690fbf5af","added_by":"auto","created_at":"2025-10-31 14:51:55","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":258113,"visible":true,"origin":"","legend":"\u003cp\u003eIn vivo fluorescence imaging of fluorescently labeled liposome constructs. Mice were imaged in supine and prone positions at 1, 2, 3, and 4 days post-injection\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-7688492/v1/5ef15b908db9e6b299f72152.png"},{"id":94870546,"identity":"d063cdc6-620a-445d-9824-fbde9e072655","added_by":"auto","created_at":"2025-10-31 14:51:55","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":586115,"visible":true,"origin":"","legend":"\u003cp\u003e(a) Immunofluorescence for CD11b (a1, a5), F4/80 (a2, a6), CCR7 (a3, a7), and CD206 (a4, a8) in implanted TCPs at 3 days after i.p. LipPBS (a1–a4) or LipClod (a5–a8); magnified regions are shown in a1′–a8′ (red dashed boxes). Scale bars: a1–a8, 400 µm; a1′–a8′, 100 µm (b) Quantification of CD11b\u003csup\u003e+\u003c/sup\u003e, F4/80\u003csup\u003e+\u003c/sup\u003e, CCR7\u003csup\u003e+\u003c/sup\u003e, and CD206\u003csup\u003e+\u003c/sup\u003e cells\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-7688492/v1/0c302cf2efc76af35b0cc1e0.png"},{"id":94870544,"identity":"afd7c0ed-edf5-493b-9144-f517a3e8fc57","added_by":"auto","created_at":"2025-10-31 14:51:55","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":320444,"visible":true,"origin":"","legend":"\u003cp\u003e(a) Expression of F4/80, iNOS, CCR7, Arg1, CD163, and CD206 in cells on implanted TCPs at 3 days after i.p. LipPBS or LipClod (b) Flow cytometry plots showing the percentage of migrated mBMSCs at 14 days after i.p. LipPBS or LipClod (c) Corresponding quantitative analysis of the flow-cytometric data\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-7688492/v1/0fade2d845714a84b1aea82d.png"},{"id":94870552,"identity":"859fa9e5-a1df-4e78-9615-822babfbd272","added_by":"auto","created_at":"2025-10-31 14:51:55","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":724515,"visible":true,"origin":"","legend":"\u003cp\u003e(a) H\u0026amp;E staining of newly formed bone at 8 weeks after injection of PBS (a1), 0.5 µg/mL M-CSF (a2), or 2 µg/mL M-CSF (a3); magnified regions are shown in red dashed boxes. Scale bars: a1–a3, 400 µm; a1′–a3′, 100 µm. Green arrows indicate newly formed bone tissue (b) Percentage of new bone in PBS, 0.5 µg/mL M-CSF, and 2 µg/mL M-CSF groups at 8 weeks after injection; *p \u0026lt; 0.05 vs PBS; #p \u0026lt; 0.05 between M-CSF groups (n = 5)\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-7688492/v1/604d915c0fc5ef48ebb60fb7.png"},{"id":94870553,"identity":"930325cb-1ab7-46da-b9c0-326e8365c9b9","added_by":"auto","created_at":"2025-10-31 14:51:55","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":623409,"visible":true,"origin":"","legend":"\u003cp\u003e(a) Immunostaining for F4/80, CCR7, and CD206 at 3 days post-injection of PBS (a1, a4, a7), 0.5 µg/mL M-CSF (a2, a5, a8), or 2 µg/mL M-CSF (a3, a6, a9). Scale bar = 100 µm (b) Quantification of F4/80\u003csup\u003e+\u003c/sup\u003e, CCR7\u003csup\u003e+\u003c/sup\u003e, and CD206\u003csup\u003e+\u003c/sup\u003e cells in each group. (c) RT-qPCR of related gene expression at 3 days post-injection; *p \u0026lt; 0.05, **p \u0026lt; 0.01, ***p \u0026lt; 0.001, ****p \u0026lt; 0.0001 vs PBS; #p \u0026lt; 0.05, ##p \u0026lt; 0.01, ###p \u0026lt; 0.001, ####p \u0026lt; 0.0001 between M-CSF groups (one-way ANOVA with Tukey’s post hoc test) (n = 5)\u0026nbsp;\u003c/p\u003e","description":"","filename":"floatimage6.png","url":"https://assets-eu.researchsquare.com/files/rs-7688492/v1/47cc8eacbef9669ae828f789.png"},{"id":94986128,"identity":"13bb69ab-c052-48e5-8581-d01f1a2bf690","added_by":"auto","created_at":"2025-11-03 06:59:51","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":343368,"visible":true,"origin":"","legend":"\u003cp\u003e(a) Fluorescence microscopy images (a1–a4) and quantification (a5) of nuclei of cells migrating to the lower chamber after mBMSCs were cultured for 16 h in a transwell system with CM collected from TCPs–BMM co-cultures. BMM densities were 0 (control), 2.5 × 10\u003csup\u003e5\u003c/sup\u003e, 5 × 10\u003csup\u003e5\u003c/sup\u003e, and 1.0 × 10\u003csup\u003e6 \u003c/sup\u003ecells per disc for a1–a4; scale bar, 200 µm. (b) Expression of migration-related genes in each group. % indicates a significant difference versus the control; * indicates a significant difference among experimental groups\u003c/p\u003e","description":"","filename":"floatimage7.png","url":"https://assets-eu.researchsquare.com/files/rs-7688492/v1/4a42855f459a91a19698d336.png"},{"id":102785386,"identity":"9746c16a-0aea-42ae-a6a7-421530fe730c","added_by":"auto","created_at":"2026-02-16 16:06:04","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4395261,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7688492/v1/96f00932-10b9-4efd-a0bd-528fe2ced1cc.pdf"},{"id":94986971,"identity":"9916ea33-11a2-4d07-98d9-4322347d4fc9","added_by":"auto","created_at":"2025-11-03 07:01:03","extension":"png","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":438167,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eScheme 1\u003c/strong\u003e Route-dependent model: i.p. LipClod limits distal depletion, triggers systemic compensation, expands peri-implant macrophages, enhances early BMSC homing, and augments TCPs-instructed osteoinduction (MIOI)\u003c/p\u003e","description":"","filename":"floatimage8.png","url":"https://assets-eu.researchsquare.com/files/rs-7688492/v1/7b39effbbc51bbf3b7496b82.png"}],"financialInterests":"No competing interests reported.","formattedTitle":"Intraperitoneal clodronate liposomes remodel the local macrophage niche and potentiate biomaterial-induced osteoinduction","fulltext":[{"header":"1 Introduction","content":"\u003cp\u003eRegeneration of critical-sized bone defects remains a major clinical challenge. While autologous bone grafting is the clinical gold standard, it is constrained by donor-site morbidity and limited availability [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Osteoinductive biomaterials that trigger de novo bone formation at ectopic sites without exogenous cells or growth factors represent a promising alternative [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. However, successful clinical translation requires a mechanistic understanding of materials-instructed osteoinduction (MIOI). Increasing evidence indicates that macrophages are early, central regulators of MIOI [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. After implantation, circulating monocytes rapidly accumulate on biomaterial surfaces, differentiate into macrophages, and polarize into distinct functional states (e.g., pro-inflammatory M1 and pro-reparative M2) in response to local cues [\u003cspan additionalcitationids=\"CR6\" citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. For instance, our previous work demonstrated that scaffold surface topography can direct macrophage polarization to promote or impede osteogenesis [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eliposomal clodronate (LipClod) is a widely used pharmacological agent that selectively depletes phagocytic macrophages, enabling causal interrogation of their roles across diverse biological processes. The magnitude and distribution of depletion depend strongly on the route of administration [\u003cspan additionalcitationids=\"CR10\" citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. Intravenous delivery produces systemic macrophage depletion, particularly in the liver and spleen, whereas subcutaneous injection confines effects locally [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. This route-dependent activity positions LipClod as a tool to modulate defined macrophage niches and dissect their contributions to tissue regeneration.\u003c/p\u003e\u003cp\u003eIn bone regeneration studies, local LipClod injection at injury or implant sites is commonly used and has been shown to consistently suppress osteogenesis by eliminating essential resident macrophages [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. By contrast, the consequences of systemic intraperitoneal (i.p.) administration remain unclear and even appear paradoxical. Sandberg et al. reported that i.p. LipClod transiently impaired bone repair, whereas Cho et al. observed increased bone mass in an orthotopic setting using the same regimen [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. These conflicting findings, derived exclusively from orthotopic models, underscore a critical knowledge gap: the impact of systemic macrophage modulation via i.p. LipClod on the distinct process of MIOI remains unknown.\u003c/p\u003e\u003cp\u003eIn this study, one kind of classical osteoinductive β-tricalcium phosphate (TCP) ceramic named TCPs was employed. Our previous studies have proved that TCPs could result in favorable bone formation at nonosseous site[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. We initially hypothesized that systemic macrophage depletion by i.p. LipClod would suppress MIOI. Unexpectedly, a single post-implantation i.p. dose markedly enhanced ectopic bone formation. This finding prompted mechanistic analyses of local cellular and molecular responses to identify the basis of the pro-osteogenic effect. Here, we reveal a route-specific immunomodulatory mechanism whereby systemic perturbation of the macrophage compartment can be exploited to improve the performance of osteoinductive biomaterials.\u003c/p\u003e"},{"header":"2 Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003e2.1 Materials and reagents\u003c/h2\u003e\u003cp\u003ePorous TCPs (4 \u0026times; 4 \u0026times; 4 mm) with submicron-scale surface topography (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003ea) were supplied by Kuros Biosciences B.V. (Bilthoven, the Netherlands). Scaffolds were sterilized by autoclaving at 121\u0026deg;C for 30 min before use. LipClod and vehicle liposomes containing phosphate-buffered saline (LipPBS) were obtained from Vrije Universiteit Amsterdam (Amsterdam, the Netherlands). All other reagents were of analytical grade or higher.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\u003ch2\u003e2.2 In vivo studies\u003c/h2\u003e\u003cdiv id=\"Sec5\" class=\"Section3\"\u003e\u003ch2\u003e2.2.1 Animals\u003c/h2\u003e\u003cp\u003eMale FVB mice (5\u0026ndash;6 weeks old) were purchased from Charles River Laboratories (Beijing, China). Mice were maintained under specific pathogen-free (SPF) conditions with ad libitum access to food and water.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec6\" class=\"Section3\"\u003e\u003ch2\u003e2.2.2 Surgical procedures\u003c/h2\u003e\u003cp\u003e All animal procedures complied with the ARRIVE guidelines and were approved by the Animal Care and Use Committee of Sichuan University. A dorsal subcutaneous implantation model was used as previously described [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Briefly, anesthesia was induced and maintained with 2% isoflurane, and buprenorphine (0.1 mg/kg) was administered subcutaneously (s.c.) for analgesia. After aseptic preparation of the dorsal skin, 2\u0026ndash;4 subcutaneous pockets were created, and one TCPs was inserted into each pocket.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec7\" class=\"Section3\"\u003e\u003ch2\u003e2.2.3 Systemic LipClod and vehicle administration\u003c/h2\u003e\u003cp\u003e1 day after implantation, mice received a single i.p. injection of LipClod at 10 \u0026micro;L/g body weight. Control animals received an equal volume of LipPBS. For biodistribution analysis, a separate cohort received a single i.p. injection of fluorescently labeled LipPBS (10 \u0026micro;L/g).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec8\" class=\"Section3\"\u003e\u003ch2\u003e2.2.4 Local M-CSF administration\u003c/h2\u003e\u003cp\u003eTo augment local macrophage numbers, we performed a validation experiment with M-CSF (PeproTech, USA) as previously described [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. Scaffolds were injected locally with M-CSF (100 \u0026micro;L; 0.5 or 2.0 \u0026micro;g/mL), whereas contralateral pockets received an equal volume of PBS as controls.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec9\" class=\"Section3\"\u003e\u003ch2\u003e2.2.5 Scaffold harvesting and processing\u003c/h2\u003e\u003cp\u003eAt predefined time points, scaffolds were explanted for analysis. For flow cytometry, explants were dissected free of surrounding tissue and enzymatically digested in RPMI containing 0.5 mg/mL Liberase TL (Sigma, Germany) at 37\u0026deg;C for 45 min with agitation. Cell suspensions were centrifuged (1000 rpm, 5 min), resuspended in PBS, and filtered through a 100 \u0026micro;m strainer to obtain single-cell suspensions. For other analyses, explants were fixed in 4% formaldehyde for histology or snap-frozen and stored at \u0026minus;\u0026thinsp;80\u0026deg;C for RNA isolation.\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec10\" class=\"Section2\"\u003e\u003ch2\u003e2.3 In vitro studies\u003c/h2\u003e\u003cdiv id=\"Sec11\" class=\"Section3\"\u003e\u003ch2\u003e2.3.1 Isolation and culture of bone marrow-derived macrophages (BMMs)\u003c/h2\u003e\u003cp\u003eDetailed methods are described in our recent work [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. In brief, complete growth medium (GM) consisting of Minimum Essential Medium α (α-MEM; Gibco, USA), 10% fetal bovine serum (FBS; Gibco, USA), and 1% (v/v) penicillin/streptomycin, supplemented with 100 ng/mL M-CSF (AF-315-02-100; PeproTech, USA), was prepared. Cells harvested from the bone marrow were then washed with PBS and cultured in GM in an incubator at 37\u0026deg;C with 5% CO2. After 24 h, the cell suspension was transferred to new culture flasks and cultured for an additional 2 days to allow BMMs to adhere. Finally, a single-cell BMM suspension was prepared by gently scraping.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec12\" class=\"Section3\"\u003e\u003ch2\u003e2.3.2 Murine bone marrow mesenchymal stem cell (mBMSC) migration assay\u003c/h2\u003e\u003cp\u003eBMMs at different densities (2.5 \u0026times; 10\u003csup\u003e5,\u003c/sup\u003e 5 \u0026times; 10\u003csup\u003e5\u003c/sup\u003e, and 1.0 \u0026times; 10\u003csup\u003e6\u003c/sup\u003e cells per disc) were seeded on TCPs. Then, 50 ng/mL M-CSF was added to the medium, and the cells and TCPs scaffolds were co-cultured for 4 days. The supernatant was collected and mixed 1:1 with GM to generate conditioned medium (CM). A 24-well Transwell system (8 \u0026micro;m pore size; Corning, USA) was used to assess mBMSC migration. Briefly, mBMSCs (CRL-12424) at 2 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e cells per well in serum-free DMEM were seeded into the upper chamber, and 500 \u0026micro;L of CM was added to the lower chamber. After 16 h, cells that migrated to the lower chamber were stained with DAPI, imaged using an inverted fluorescence microscope, and quantified in ImageJ (n\u0026thinsp;=\u0026thinsp;3).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec13\" class=\"Section3\"\u003e\u003ch2\u003e2.3.3 mBMSC osteogenic differentiation assay\u003c/h2\u003e\u003cp\u003eTo assess osteogenic differentiation, mBMSCs (2 \u0026times; 10\u003csup\u003e4\u003c/sup\u003e) were cultured for 7 days in CM prepared as above; cells maintained in standard complete medium served as controls. After 7 days, cells were harvested for RT-qPCR (n\u0026thinsp;=\u0026thinsp;4).\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003e2.4 Evaluation\u003c/h2\u003e\u003cdiv id=\"Sec15\" class=\"Section3\"\u003e\u003ch2\u003e2.4.1 Histological analysis\u003c/h2\u003e\u003cp\u003eFixed samples were processed for either undecalcified or decalcified sectioning. For undecalcified sections, samples were dehydrated through graded ethanol, embedded in polymethyl methacrylate, sectioned at 10\u0026ndash;20 \u0026micro;m, and stained with methylene blue and basic fuchsin. For decalcified sections, samples were treated in 0.5 M EDTA for 10\u0026ndash;14 days, dehydrated, and embedded in paraffin; 4\u0026ndash;7 \u0026micro;m sections were cut and stained with Masson\u0026rsquo;s trichrome. All sections were imaged with a light microscope (Leica, Germany).\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec16\" class=\"Section3\"\u003e\u003ch2\u003e2.4.2 In vivo fluorescence imaging\u003c/h2\u003e\u003cp\u003eMice receiving fluorescently labeled liposomes were anesthetized with 2% isoflurane and imaged at 1, 2, 3, and 4 days post-injection in supine and prone positions using an IVIS Lumina III system (PerkinElmer, USA)\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec17\" class=\"Section3\"\u003e\u003ch2\u003e2.4.3 Immunohistochemistry (IHC)\u003c/h2\u003e\u003cp\u003eDeparaffinized sections underwent antigen retrieval in citrate buffer (95\u0026deg;C, 5 min). Endogenous peroxidase activity was quenched and non-specific binding was blocked with goat serum. Sections were incubated overnight at 4\u0026deg;C with primary antibodies against F4/80 (1:100; Abcam, ab100790), CCR7 (1:50; Novus Biologicals, NB100-712), CD163 (1:200; Abcam, ab182422), or CD206 (HuaBio, JF0953). After incubation with appropriate secondary antibodies, signals were developed using a DAB chromogen kit and counterstained with hematoxylin.\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec18\" class=\"Section3\"\u003e\u003ch2\u003e2.4.4 Flow cytometry\u003c/h2\u003e\u003cp\u003eSingle-cell suspensions were permeabilized using a Foxp3/Transcription Factor Staining Buffer Set (eBioscience) and incubated with fluorochrome-conjugated monoclonal antibodies at 4\u0026deg;C for 40 min. Mesenchymal stem cells (MSCs) were gated as CD29\u003csup\u003e+\u003c/sup\u003e/CD44\u003csup\u003e+\u003c/sup\u003e/CD90\u003csup\u003e+\u003c/sup\u003e and CD34\u003csup\u003e\u0026minus;\u003c/sup\u003e/CD45\u003csup\u003e\u0026minus;\u003c/sup\u003e. Data were acquired on a ZE5 Cell Analyzer (Bio-Rad) and analyzed in Kaluza (n\u0026thinsp;=\u0026thinsp;4) [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e].\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec19\" class=\"Section3\"\u003e\u003ch2\u003e2.4.5 RT-qPCR\u003c/h2\u003e\u003cp\u003eSamples were thoroughly pulverized in a glass homogenizer at 4\u0026deg;C. Total RNA was isolated using TRIzol reagent (Thermo Fisher Scientific, USA) according to the manufacturer\u0026rsquo;s instructions. RNA concentration was measured on a NanoDrop spectrophotometer (Thermo Fisher Scientific, USA). cDNA was synthesized using the RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific, USA) following the manufacturer\u0026rsquo;s instructions. qPCR was performed on an ABI StepOnePlus\u0026trade; Real-Time PCR System with an initial denaturation at 95\u0026deg;C for 10 min, followed by 40 cycles of 95\u0026deg;C for 30 s, 60\u0026deg;C for 1 min, and 72\u0026deg;C for 1 min. GAPDH was chosen as the housekeeping gene for normalization. BSP was initially assessed to evaluate variations in TCPs-instructed osteogenesis under the influence of LipClod or LipPBS. For macrophage polarization, expression of F4/80 (a pan-macrophage marker), CCR7/iNOS (M1 markers), and Arg1/CD163/CD206 (M2 markers) was evaluated; BSP was also quantified. For mBMSC osteogenic differentiation, gene expression of ALP, OCN, OPN, Runx2, Osterix, and Col1a1 was investigated. For each gene, 4\u0026ndash;5 samples were analyzed, and each sample was assayed in triplicate. Relative gene expression was calculated using the 2^\u0026minus;ΔΔCt method.\u003c/p\u003e\u003c/div\u003e\u003c/div\u003e\u003cdiv id=\"Sec20\" class=\"Section2\"\u003e\u003ch2\u003e2.5 Statistical analysis\u003c/h2\u003e\u003cp\u003eData are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation (SD). Statistical significance was determined using Student\u0026rsquo;s t test for two-group comparisons or one-way analysis of variance (ANOVA) with Tukey\u0026rsquo;s post hoc test for multiple comparisons. A p value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was considered statistically significant (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05; *p\u0026thinsp;\u0026lt;\u0026thinsp;0.01; **p\u0026thinsp;\u0026lt;\u0026thinsp;0.001; ***p\u0026thinsp;\u0026lt;\u0026thinsp;0.0001).\u003c/p\u003e\u003c/div\u003e"},{"header":"3 Results","content":"\u003cdiv id=\"Sec22\" class=\"Section2\"\u003e\u003ch2\u003e3.1 i.p. LipClod administration enhances TCPs-instructed ectopic osteogenesis\u003c/h2\u003e\u003cp\u003eEight weeks post-implantation, significant osteogenesis was observed in the LipPBS control group (Fig.\u0026nbsp;1b1, 1c1, 1c1\u0026prime;), confirming the favorable osteoinductive properties of the TCPs scaffold. Compared with LipPBS, LipClod treatment significantly promoted new bone formation (Fig.\u0026nbsp;1b2, 1c2, 1c2\u0026prime;, 1d), indicating that i.p. LipClod enhances TCPs-instructed osteoinduction. Furthermore, RT-qPCR showed a significant upregulation of bone sialoprotein (BSP) transcripts following i.p. LipClod administration, a molecule implicated in the early-stage formation of apatite crystals [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e].\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec23\" class=\"Section2\"\u003e\u003ch2\u003e3.2 Biodistribution of liposomes following i.p. injection\u003c/h2\u003e\u003cp\u003eAs revealed by fluorescence imaging (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e), following i.p. injection, most liposomes remained localized at the injection site throughout the observation period, with only a small fraction reaching the distal implantation site via the circulation. The fluorescence intensity at the implantation site peaked at day 3 post-injection and subsequently declined, suggesting clearance of liposomes, potentially via the kidneys.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec24\" class=\"Section2\"\u003e\u003ch2\u003e3.3 i.p. LipClod injection modulates macrophage recruitment and polarization\u003c/h2\u003e\u003cp\u003eAt day 3 post-injection, immunostaining (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e) revealed more CD11b\u003csup\u003e+\u003c/sup\u003e, F4/80\u003csup\u003e+\u003c/sup\u003e, and CCR7\u003csup\u003e+\u003c/sup\u003e cells in the LipClod group than in LipPBS, whereas CD206\u003csup\u003e+\u003c/sup\u003e (M2) cells did not differ between groups. At the transcript level (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea), LipClod increased F4/80 and iNOS (M1) expression. Despite comparable CD206\u003csup\u003e+\u003c/sup\u003e counts by IHC, Arg1 and CD206 transcripts were also elevated in the LipClod group [\u003cspan additionalcitationids=\"CR25\" citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e].\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec25\" class=\"Section2\"\u003e\u003ch2\u003e3.4 i.p. LipClod administration promotes mBMSC migration\u003c/h2\u003e\u003cp\u003eTo assess whether i.p. LipClod affects mBMSC migration, we quantified migrated cells using flow cytometry in Transwell assays. Compared with LipPBS controls (20.28%), i.p. LipClod increased the proportion of migrated mBMSCs to 29.48% (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb, \u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ec).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec26\" class=\"Section2\"\u003e\u003ch2\u003e3.5 Local M-CSF injection augments osteogenesis and modulates macrophage behavior\u003c/h2\u003e\u003cp\u003eHistology showed that local M-CSF enhanced ectopic osteogenesis in a concentration-dependent manner (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea); the higher dose (2 \u0026micro;g/mL) produced significantly more new bone than control (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eb). Concordantly, IHC and RT-qPCR demonstrated dose-dependent recruitment of F4/80\u003csup\u003e+\u003c/sup\u003e macrophages (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e). Notably, increased recruitment coincided with fewer CCR7\u003csup\u003e+\u003c/sup\u003e M1 macrophages, suggesting that M-CSF also modulates polarization [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. RT-qPCR further showed a negative correlation between CCR7 transcripts and M-CSF dose, whereas CD206 (M2) expression increased with M-CSF concentration.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec27\" class=\"Section2\"\u003e\u003ch2\u003e3.6 Effect of macrophage population on the migration and osteogenic differentiation of mBMSCs\u003c/h2\u003e\u003cp\u003eThe Transwell migration assay demonstrated that CM from co-cultures with higher macrophage populations significantly promoted migration of murine bone marrow\u0026ndash;derived mBMSCs (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ea). In contrast, for osteogenic differentiation, CM from any macrophage density did not significantly alter expression of ALP, OCN, or Osterix compared with controls (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003eb). Furthermore, CM treatment significantly decreased expression of OPN, Col1a1, and Runx2 relative to controls. Among the different macrophage-density groups, a significant difference was observed only for Runx2 between the 2.5 \u0026times; 10⁵ and 1.0 \u0026times; 10⁶ cells per disc groups.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e"},{"header":"4 Discussion","content":"\u003cp\u003eConventionally, macrophage depletion using LipClod is considered detrimental to MIOI [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. In contrast, we found that i.p. LipClod promoted TCPs-instructed ectopic osteogenesis, indicating a route-dependent host response.\u003c/p\u003e\u003cp\u003eLiposome biodistribution tracking offers a mechanistic explanation. After i.p. injection, most liposomes (LipClod) remained within the peritoneal cavity and only a minor fraction reached distal implants, consistent with liver and spleen accumulation and low plasma levels reported by Ylitalo [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. Consequently, drug levels at the implant are too low and transient for durable macrophage depletion, potentially modulating rather than ablating local macrophages.\u003c/p\u003e\u003cp\u003eAt the implant site, we observed increased CD11b\u003csup\u003e+\u003c/sup\u003e and F4/80\u003csup\u003e+\u003c/sup\u003e cells with broad upregulation of M1 (CCR7, iNOS) and M2 (CD163, Arg1, CD206) markers, indicating robust monocyte recruitment and differentiation and a mixed pro-inflammatory/reparative milieu.\u003c/p\u003e\u003cp\u003eThese changes align with a systemic compensation model. i.p. LipClod depletes macrophages in peritoneal and omental niches within days [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e], prompting increased bone marrow monocyte output and peripheral mobilization; newly generated cells home to injured scaffolds in response to inflammatory cues [\u003cspan additionalcitationids=\"CR31\" citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Consistently, long-term i.p. LipClod treatment increased tibial trabecular bone mass and potentiated intermittent parathyroid hormone responses with enrichment of CD68\u003csup\u003e+\u003c/sup\u003e/CD163\u003csup\u003e+\u003c/sup\u003e myeloid cells and upregulation of osteogenic genes (Wnt3a, Wnt10b, Tgfb1) [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. In a tooth-extraction model, site-dependent effects were observed: tibial BV/TV rose by 81% (day 7) and 152% (day 14) versus PBS, whereas oral socket bone fill increased by ~\u0026thinsp;7% (day 14), with steady-state oral bone unchanged [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. Moreover, i.p. LipClod directly affects bone marrow monocytes, decreasing mature and increasing immature subsets for ~\u0026thinsp;3\u0026ndash;5 days [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Thus, any limited local depletion is rapidly offset by influx and local proliferation of newly recruited macrophages.\u003c/p\u003e\u003cp\u003eAn expanded macrophage population appears to promote osteogenesis primarily by enhancing MSC recruitment [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. CM from pro-inflammatory or repair-associated macrophages stimulates MSC recruitment, with stronger effects for pro-inflammatory CM\u0026mdash;likely via factors such as OSM, TNF-α, and IL-10 that drive MSC elongation [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]; M1-like macrophages are particularly important for this process [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. In our in vitro experiments, CM from high-density macrophage cultures significantly promoted MSC migration, whereas it either failed to increase or even decreased expression of osteogenic markers. This observation is consistent with previous evidence showing that pro-inflammatory macrophage CM favors MSC migration, whereas anti-inflammatory factors are more closely associated with osteogenic differentiation [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Moreover, mechanistic studies have demonstrated that macrophage-derived chemokines such as CCL5, CCL2, and IL-8 can drive a\u0026thinsp;~\u0026thinsp;5\u0026ndash;9-fold increase in MSC chemotaxis through JNK signaling [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Taken together, these findings and our own results support a model in which i.p. LipClod generates an early, chemokine-rich microenvironment that efficiently recruits host MSCs, thereby establishing the cellular basis for subsequent osteogenesis.\u003c/p\u003e\u003cp\u003eTo directly test the \u0026ldquo;numbers matter\u0026rdquo; hypothesis, local injections of M-CSF increased macrophage infiltration and enhanced bone formation in a dose-dependent manner [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. M-CSF also biased polarization toward an M2 phenotype and underscored that macrophage abundance and timing, rather than any single polarization state, govern the pro-osteogenic niche [40].\u003c/p\u003e\u003cp\u003eTogether, these findings support a route-dependent model of clearance followed by compensation: i.p. delivery confines most liposomes to the peritoneal compartment, limiting distal depletion; transient loss of peritoneal and omental macrophages drives systemic myelopoiesis and monocyte recruitment to implants; the ensuing early, mixed M1/M2, chemokine-rich milieu primarily enhances MSC homing with limited direct effects on osteogenic differentiation. This framework predicts that attenuating early inflammatory cues (24\u0026ndash;72 h) will blunt MSC homing and weaken MIOI, whereas interventions at later stages will have smaller effects; it also highlights dose and route of administration as actionable levers to engineer osteoinductive immunity.\u003c/p\u003e"},{"header":"5 Conclusion","content":"\u003cp\u003eThis study demonstrates that i.p. delivery of LipClod paradoxically enhances TCPs-instructed ectopic osteogenesis by increasing peri-implant macrophage abundance. We propose that this systemic stimulus elicits a compensatory host response that accelerates production and preferential homing of monocyte\u0026ndash;macrophage precursors to the implant site (Scheme \u003cspan refid=\"Sch1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). This, in turn, enhances recruitment of MSCs\u0026mdash;crucial for osteogenesis\u0026mdash;likely via pro-inflammatory cytokines. Collectively, our findings present a route-specific strategy to potentiate MIOI by modulating systemic host responses and provide evidence that macrophage abundance is a key determinant in osteoimmunology. These insights rationalize the development of immunomodulatory biomaterial strategies to enhance bone regeneration and may help accelerate clinical translation.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eFunding:\u0026nbsp;\u003c/strong\u003eThis research was supported by grants from the National Natural Science Foundation of China (Grant No. 82371013) and the Research and Development Program of the West China Hospital of Stomatology, Sichuan University (Grant No. RD-03-202306).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests:\u003c/strong\u003e The authors declare no competing interests.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval:\u003c/strong\u003e All animal procedures were approved by the Animal Care and Use Committee of Sichuan University (Approval No. WCHSIRB-D-2024-525).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to participate:\u003c/strong\u003e Not applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication:\u0026nbsp;\u003c/strong\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e: All data supporting the findings of this study are included in the article. Raw datasets are available from the corresponding author upon reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; contributions:\u0026nbsp;\u003c/strong\u003eWei Cao and Zhiqiao Hu performed the investigation and drafted the original manuscript. Xiaodong Guo contributed to methodology and investigation. Mingzheng Li, Zhangling Nie, and Chengen Li performed investigation. Wenting Qi and Yue Wang performed histological assessments. Yu Xiao led conceptualization, provided supervision, and handled writing\u0026mdash;review and editing. Chongyun Bao secured funding (funding acquisition). All authors commented on previous versions of the manuscript, and all authors read and approved the final manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eRoddy E, DeBaun MR, Daoud-Gray A, Yang YP, Gardner MJ (2018) Treatment of critical-sized bone defects: clinical and tissue engineering perspectives. Eur J Orthop Surg Traumatol 28:351\u0026ndash;362. https://doi.org/10.1007/s00590-017-2063-0\u003c/li\u003e\n\u003cli\u003eSchmidt AH (2021) Autologous bone graft: is it still the gold standard? Injury 52(Suppl 2):18\u0026ndash;22. https://doi.org/10.1016/j.injury.2021.01.043\u003c/li\u003e\n\u003cli\u003eBai H, Guo X, Tan Y, Wang Y, Feng J, Lei K, Liu X, Xiao Y, Bao C (2022) Hypoxia-inducible factor-1 signaling pathway in macrophage-involved angiogenesis in materials-instructed osteoinduction. J Mater Chem B 10:6483\u0026ndash;6495. https://doi.org/10.1039/d2tb00811d\u003c/li\u003e\n\u003cli\u003eBohner M, Miron RJ (2019) A proposed mechanism for material-induced heterotopic ossification. Mater Today 22:132\u0026ndash;141. https://doi.org/10.1016/j.mattod.2018.10.036\u003c/li\u003e\n\u003cli\u003ePajarinen J, Lin T, Gibon E, Kohno Y, Maruyama M, Nathan K, Lu L, Yao Z, Goodman SB (2019) Mesenchymal stem cell\u0026ndash;macrophage crosstalk and bone healing. Biomaterials 196:80\u0026ndash;89. https://doi.org/10.1016/j.biomaterials.2017.12.025\u003c/li\u003e\n\u003cli\u003eLee J, Byun H, Madhurakkat Perikamana SK, Lee S, Shin H (2019) Current advances in immunomodulatory biomaterials for bone regeneration. Adv Healthc Mater 8:e1801106. https://doi.org/10.1002/adhm.201801106\u003c/li\u003e\n\u003cli\u003eNewman H, Shih YV, Varghese S (2021) Resolution of inflammation in bone regeneration: from understandings to therapeutic applications. Biomaterials 277:121114. https://doi.org/10.1016/j.biomaterials.2021.121114\u003c/li\u003e\n\u003cli\u003eLi M, Guo X, Qi W, Wu Z, de Bruijn JD, Xiao Y, Bao C, Yuan H (2020) Macrophage polarization plays roles in bone formation instructed by calcium phosphate ceramics. J Mater Chem B 8:1863\u0026ndash;1877. https://doi.org/10.1039/c9tb02932j\u003c/li\u003e\n\u003cli\u003eAlexander KA, Chang MK, Maylin ER, Kohler T, Muller R, Wu AC, van Rooijen N, Sweet MJ, Hume DA, Raggatt LJ, Pettit AR (2011) Osteal macrophages promote in vivo intramembranous bone healing in a mouse tibial injury model. J Bone Miner Res 26:1517\u0026ndash;1532. https://doi.org/10.1002/jbmr.354\u003c/li\u003e\n\u003cli\u003evan Rooijen N, Hendrikx E (2010) Liposomes for specific depletion of macrophages from organs and tissues. Methods Mol Biol 605:189\u0026ndash;203. https://doi.org/10.1007/978-1-60327-360-2_13\u003c/li\u003e\n\u003cli\u003eMcCloskey E, Paterson AH, Powles T, Kanis JA (2020) Clodronate. Bone 143:115715. https://doi.org/10.1016/j.bone.2020.115715\u003c/li\u003e\n\u003cli\u003eMichalski MN, Zweifler LE, Sinder BP, Koh AJ, Yamashita J, Roca H, McCauley LK (2019) Clodronate-loaded liposome treatment has site-specific skeletal effects. J Dent Res 98:459\u0026ndash;467. https://doi.org/10.1177/0022034518821685\u003c/li\u003e\n\u003cli\u003eOkada E, Nakata H, Yamamoto M, Kasugai S, Kuroda S (2018) Indirect osteoblast differentiation by liposomal clodronate. J Cell Mol Med 22:1127\u0026ndash;1137. https://doi.org/10.1111/jcmm.13366\u003c/li\u003e\n\u003cli\u003eDai B, Xu J, Li X, Huang L, Hopkins C, Wang H, Yao H, Mi J, Zheng L, Wang J, Tong W, Chow DH-K, Li Y, He X, Hu P, Chen Z, Zu H, Li Y, Yao Y, Jiang Q, Qin L (2022) Macrophages in epididymal adipose tissue secrete osteopontin to regulate bone homeostasis. Nat Commun 13:427. https://doi.org/10.1038/s41467-021-27683-w\u003c/li\u003e\n\u003cli\u003eDavison NL, Gamblin AL, Layrolle P, Yuan H, de Bruijn JD, Barrere-de Groot F (2014) Liposomal clodronate inhibition of osteoclastogenesis and osteoinduction by submicrostructured beta-tricalcium phosphate. Biomaterials 35:5088\u0026ndash;5097. https://doi.org/10.1016/j.biomaterials.2014.03.013\u003c/li\u003e\n\u003cli\u003eSandberg OH, Tatting L, Bernhardsson ME, Aspenberg P (2017) Temporal role of macrophages in cancellous bone healing. Bone 101:129\u0026ndash;133. https://doi.org/10.1016/j.bone.2017.04.004\u003c/li\u003e\n\u003cli\u003eCho SW, Soki FN, Koh AJ, Eber MR, Entezami P, Park SI, van Rooijen N, McCauley LK (2014) Osteal macrophages support physiologic skeletal remodeling and anabolic actions of parathyroid hormone in bone. Proc Natl Acad Sci U S A 111:1545\u0026ndash;1550. https://doi.org/10.1073/pnas.1315153111\u003c/li\u003e\n\u003cli\u003eGuo X, Li M, Qi W, Bai H, Nie Z, Hu Z, Xiao Y, de Bruijn JD, Bao C, Yuan H (2021) Serial cellular events in bone formation initiated by calcium phosphate ceramics. Acta Biomater 134:730\u0026ndash;743. https://doi.org/10.1016/j.actbio.2021.07.037\u003c/li\u003e\n\u003cli\u003eNie Z, Hu Z, Guo X, Xiao Y, Liu X, de Bruijn JD, Bao C, Yuan H (2023) Genesis of osteoclasts on calcium phosphate ceramics and their role in material-induced bone formation. Acta Biomater 157:625\u0026ndash;638. https://doi.org/10.1016/j.actbio.2022.11.005\u003c/li\u003e\n\u003cli\u003eO\u0026rsquo;Brien EM, Risser GE, Spiller KL (2019) Sequential drug delivery to modulate macrophage behavior and enhance implant integration. Adv Drug Deliv Rev 149\u0026ndash;150:85\u0026ndash;94. https://doi.org/10.1016/j.addr.2019.05.005\u003c/li\u003e\n\u003cli\u003eWexler SA, Donaldson C, Kendall PD, Rice C, Bradley B, Hows JM (2003) Adult bone marrow is a rich source of human mesenchymal \u0026lsquo;stem\u0026rsquo; cells but umbilical cord and mobilized adult blood are not. Br J Haematol 121:368\u0026ndash;374. https://doi.org/10.1046/j.1365-2141.2003.04284.x\u003c/li\u003e\n\u003cli\u003eMalaval L, Wade-Gueye NM, Boudiffa M, Fei J, Zirngibl R, Chen F, Laroche N, Roux JP, Burt-Pichat B, Duboeuf F, Boivin G, Jurdic P, Lafage-Proust MH, Amedee J, Vico L, Rossant J, Aubin JE (2008) Bone sialoprotein plays a functional role in bone formation and osteoclastogenesis. J Exp Med 205:1145\u0026ndash;1153. https://doi.org/10.1084/jem.20071294\u003c/li\u003e\n\u003cli\u003eKruger TE, Miller AH, Godwin AK, Wang J (2014) Bone sialoprotein and osteopontin in bone metastasis of osteotropic cancers. Crit Rev Oncol Hematol 89:330\u0026ndash;341. https://doi.org/10.1016/j.critrevonc.2013.08.013\u003c/li\u003e\n\u003cli\u003eTsukasaki M, Takayanagi H (2019) Osteoimmunology: evolving concepts in bone\u0026ndash;immune interactions in health and disease. Nat Rev Immunol 19:626\u0026ndash;642. https://doi.org/10.1038/s41577-019-0178-8\u003c/li\u003e\n\u003cli\u003eOkamoto K, Nakashima T, Shinohara M, Negishi-Koga T, Komatsu N, Terashima A, Sawa S, Nitta T, Takayanagi H (2017) Osteoimmunology: the conceptual framework unifying the immune and skeletal systems. Physiol Rev 97:1295\u0026ndash;1349. https://doi.org/10.1152/physrev.00036.2016\u003c/li\u003e\n\u003cli\u003eZhao Q, Liu X, Yu C, Xiao Y (2022) Macrophages and bone marrow-derived mesenchymal stem cells work in concert to promote fracture healing: a brief review. DNA Cell Biol 41:276\u0026ndash;284. https://doi.org/10.1089/dna.2021.0869\u003c/li\u003e\n\u003cli\u003eMonkkonen J, Ylitalo P (1990) The tissue distribution of clodronate (dichloromethylene bisphosphonate) in mice: effects of vehicle and route of administration. Eur J Drug Metab Pharmacokinet 15:239\u0026ndash;243. https://doi.org/10.1007/BF03190210\u003c/li\u003e\n\u003cli\u003eBiewenga J, van der Ende MB, Krist LFG, et al. (1995) Macrophage depletion in the rat after i.p. administration of liposome-encapsulated clodronate: depletion kinetics and accelerated repopulation of peritoneal and omental macrophages by administration of Freund\u0026rsquo;s adjuvant. Cell Tissue Res 280:189\u0026ndash;196. https://doi.org/10.1007/BF00304524\u003c/li\u003e\n\u003cli\u003eBu L, Gao M, Qu S, Liu D (2013) Intraperitoneal injection of clodronate liposomes eliminates visceral adipose tissue macrophages. AAPS J 15:1001\u0026ndash;1011. https://doi.org/10.1208/s12248-013-9501-7\u003c/li\u003e\n\u003cli\u003eLouwe PA, Badiola Gomez L, Webster H, Perona-Wright G, Bain CC, Forbes SJ, Jenkins SJ (2021) Recruited macrophages that colonize the post-inflammatory peritoneal niche convert into functionally divergent resident cells. Nat Commun 12:1770. https://doi.org/10.1038/s41467-021-21778-0\u003c/li\u003e\n\u003cli\u003eKaur S, Sehgal A, Wu AC, et al. (2021) Stable colony-stimulating factor 1 fusion protein treatment increases hematopoietic stem cell pool and enhances their mobilisation in mice. J Hematol Oncol 14:3. https://doi.org/10.1186/s13045-020-00997-w\u003c/li\u003e\n\u003cli\u003eOza D, Ivich F, Pace J, Yu M, Niedre M, Amiji M (2024) Lipid nanoparticle\u0026ndash;encapsulated large peritoneal macrophages migrate to the lungs via the systemic circulation in a model of clodronate-mediated lung-resident macrophage depletion. Theranostics 14(6):2526\u0026ndash;2543. https://doi.org/10.7150/thno.91062\u003c/li\u003e\n\u003cli\u003eMichalski MN, McCauley LK (2019) Clodronate-loaded liposome treatment has site-specific skeletal effects. JBMR Plus 3(2):e10245. https://doi.org/10.1002/jbm4.10245\u003c/li\u003e\n\u003cli\u003eSong G, Habibovic P, Bao C, Hu J, van Blitterswijk CA, Yuan H, Chen W, Xu HH (2013) The homing of bone marrow MSCs to non-osseous sites for ectopic bone formation induced by osteoinductive calcium phosphate. Biomaterials 34:2167\u0026ndash;2176. https://doi.org/10.1016/j.biomaterials.2012.12.010\u003c/li\u003e\n\u003cli\u003eVall\u0026eacute;s G, Bensiamar F, Maestro-Paramio L, Garc\u0026iacute;a-Rey E, Vilaboa N, Salda\u0026ntilde;a L (2020) Influence of inflammatory conditions provided by macrophages on osteogenic ability of mesenchymal stem cells. Stem Cell Res Ther 11:57. https://doi.org/10.1186/s13287-020-1578-1Wasnik \u003c/li\u003e\n\u003cli\u003eS, Rundle CH, Baylink DJ, Yazdi MS, Carreon EE, Xu Y, Qin X, Lau KW, Tang X (2018) 1,25-Dihydroxyvitamin D suppresses M1 macrophages and promotes M2 differentiation at bone injury sites. JCI Insight 3:e98773. https://doi.org/10.1172/jci.insight.98773\u003c/li\u003e\n\u003cli\u003eAnton K, Banerjee D, Glod J (2012) Macrophage-associated mesenchymal stem cells assume an activated, migratory, pro-inflammatory phenotype with increased IL-6 and CXCL10 secretion. PLoS One 7:e35036. https://doi.org/10.1371/journal.pone.0035036\u003c/li\u003e\n\u003cli\u003eStarlinger J, Sarahrudi K, Kecht M, Koerbler F, Pietschmann P, Aharinejad S (2021) The influence of M-CSF on fracture healing in a mouse model. Sci Rep 11:22326. https://doi.org/10.1038/s41598-021-01673-w\u003c/li\u003e\n\u003cli\u003eVogel DYS, Glim JE, Stavenuiter AWD, Breur M, Heijnen P, Amor S, Dijkstra CD, Beelen RHJ (2014) Human macrophage polarization in vitro: maturation and activation methods compared. Immunobiology 219:695\u0026ndash;703. https://doi.org/10.1016/j.imbio.2014.05.002\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Scheme 1","content":"\u003cp\u003eScheme 1 is available in the Supplementary Files section.\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"molecular-biology-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mole","sideBox":"Learn more about [Molecular Biology Reports](https://www.springer.com/journal/11033)","snPcode":"11033","submissionUrl":"https://submission.nature.com/new-submission/11033/3","title":"Molecular Biology Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Osteogenesis, Ectopic, Macrophages, Mesenchymal Stem Cells, Cell Movement, Clodronic Acid, Immunomodulation","lastPublishedDoi":"10.21203/rs.3.rs-7688492/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7688492/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e\u003cp\u003eOsteoinductive biomaterials hold promise for repairing critical-size bone defects, yet the mechanisms underlying materials-instructed osteoinduction (MIOI) remain unclear. Macrophages are central, and local liposomal clodronate (LipClod) typically suppresses osteogenesis. Whether systemic LipClod modulates MIOI is unknown. We investigated the effect of intraperitoneal (i.p.) LipClod on ectopic osteoinduction.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e\u003cp\u003eMale FVB mice received subcutaneous β-tricalcium phosphate (TCPs) scaffolds and a single i.p. LipClod dose 1 day after implantation; vehicle liposomes served as controls (n\u0026thinsp;=\u0026thinsp;6 per group; 10 \u0026micro;L/g). Recombinant macrophage colony-stimulating factor (M-CSF; 0.5 or 2 \u0026micro;g/mL; 100 \u0026micro;L per pocket) was injected locally, with contralateral pockets receiving PBS. Outcomes were assessed by histology, immunohistochemistry, RT-qPCR, flow cytometry, and Transwell assays of macrophage-conditioned media on bone marrow mesenchymal stem cell (BMSC) migration and osteogenic genes.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e\u003cp\u003ei.p. LipClod enhanced ectopic bone formation at 8 weeks, supported by histology and increased bone sialoprotein transcripts. Rather than depleting macrophages, it expanded monocyte\u0026ndash;macrophage at the implant site and increased BMSC recruitment. Macrophage-conditioned media promoted BMSC migration in vitro. Local M-CSF further boosted macrophage infiltration, favored reparative polarization, and augmented osteogenesis.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e\u003cp\u003eSystemic LipClod augments MIOI by eliciting compensatory expansion of local macrophages that enhances early BMSC trafficking. Carefully calibrated systemic macrophage-targeted interventions may improve osteoinductive biomaterial performance.\u003c/p\u003e","manuscriptTitle":"Intraperitoneal clodronate liposomes remodel the local macrophage niche and potentiate biomaterial-induced osteoinduction","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-10-31 14:51:51","doi":"10.21203/rs.3.rs-7688492/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-11-20T15:31:07+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-11-13T14:39:56+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"2117350116283073685136960099129551390","date":"2025-11-10T15:48:07+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-10-21T12:51:06+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-09-24T07:23:25+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-09-24T07:23:10+00:00","index":"","fulltext":""},{"type":"submitted","content":"Molecular Biology Reports","date":"2025-09-23T01:32:34+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"molecular-biology-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mole","sideBox":"Learn more about [Molecular Biology Reports](https://www.springer.com/journal/11033)","snPcode":"11033","submissionUrl":"https://submission.nature.com/new-submission/11033/3","title":"Molecular Biology Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"3e9a88e7-7ea4-4d33-b0e5-e9c8efb661ff","owner":[],"postedDate":"October 31st, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2026-02-16T16:03:22+00:00","versionOfRecord":{"articleIdentity":"rs-7688492","link":"https://doi.org/10.1007/s11033-026-11533-3","journal":{"identity":"molecular-biology-reports","isVorOnly":false,"title":"Molecular Biology Reports"},"publishedOn":"2026-02-09 15:58:48","publishedOnDateReadable":"February 9th, 2026"},"versionCreatedAt":"2025-10-31 14:51:51","video":"","vorDoi":"10.1007/s11033-026-11533-3","vorDoiUrl":"https://doi.org/10.1007/s11033-026-11533-3","workflowStages":[]},"version":"v1","identity":"rs-7688492","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7688492","identity":"rs-7688492","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}