Abstract

Background

   Bone fracture regeneration poses significant clinical challenges due to
   complications such as delayed healing, nonunion, and the limitations of
   current treatments.

Objective

   This study introduces a novel therapeutic approach utilizing biomimetic
   nanogels to silence the Ccl4 gene, aiming to promote bone repair by
   regulating macrophage polarization.

Methods

   The nanogels, composed of tannic acid (TA) and small interfering RNA
   (siRNA), were designed for targeted gene delivery.

Results

   In vitro findings indicate that siRNA-mediated Ccl4 reduction
   significantly improves M2 macrophage polarization, which, in turn,
   promotes osteogenic differentiation of bone marrow-derived mesenchymal
   stem cells. Increased expression of osteogenic markers and enhanced
   mineral deposition were observed. The nanogels demonstrated optimal
   particle size, stability, and cellular uptake, and biocompatibility
   assays confirmed their non-toxicity.

Conclusion

   This study underscores the potential of targeted siRNA delivery in
   modulating immune responses to enhance bone regeneration, offering
   promising treatment options for complex bone healing scenarios.

   Keywords: Bone fracture healing, Macrophage polarization, siRNA
   therapy, Biomimetic nanogels, Osteogenic differentiation

Highlights

     * •
       Biomimetic nanogels enhance osteogenesis by targeting macrophages.
     * •
       SiRNA-modulated M2 macrophages promote BMSC osteogenic
       differentiation.
     * •
       MTA@SiCcl4 nanogels demonstrate high biocompatibility and stability
       in vitro.
     * •
       MTA@SiCcl4 nanogels exhibit superior cell-targeting capabilities.
     * •
       This approach offers a novel therapeutic strategy for complex bone
       healing.

1. Introduction

   Bone fractures pose a substantial health and economic burden, with an
   increasing prevalence due to the aging global population [[31][1],
   [32][2], [33][3]]. Despite medical advancements, a significant subset
   of fractures heal poorly, leading to substantial patient distress and
   societal costs [[34][4], [35][5], [36][6]]. Traditional treatments,
   such as autologous bone grafts, are limited by material availability,
   and the use of bone morphogenetic proteins (BMPs) carries significant
   risks, including potential carcinogenic effects and inflammatory
   reactions [[37][7], [38][8], [39][9]]. The challenge lies in the
   development of a safe and effective treatment for difficult-to-heal
   fractures.

   Effective bone healing is contingent upon the synergistic interplay
   between macrophages and BMSCs. Macrophage polarization, which is
   essential for BMSC differentiation, is a critical regulatory factor in
   this process. M1 macrophages initiate immune responses, while M2
   macrophages facilitate tissue repair by promoting BMSC osteogenesis
   [[40]10]. Recognized increasingly as crucial to bone healing, M2
   macrophages were the focus of this study, where we identified Ccl4 as a
   key gene influencing their polarization through RNA-seq analysis and in
   vitro experiments. Our study focused on M2 macrophages, which were
   identified as key to bone healing, and we targeted the Ccl4 gene, which
   influences their polarization, using RNA-seq and in vitro assays. siRNA
   holds significant therapeutic potential due to its ability to induce
   specific gene silencing [[41][11], [42][12], [43][13]]. Subsequently,
   we developed siRNA to modulate this gene, aiming to enhance BMSC
   osteogenic potential and improve bone healing.

   Despite its advantages, siRNA's clinical application is hindered by
   delivery challenges, including susceptibility to degradation and
   cellular membrane impermeability [[44]14,[45]15]. To overcome these, we
   developed TA-siRNA nanogels, which protect siRNA from degradation.
   Known for its anti-inflammatory, antibacterial, and anticancer
   properties, TA has broad biomedical applications [[46]16]. To
   facilitate targeted delivery, the application of mimicking RAW cell
   membranes which employ homologous targeting to specifically deliver
   drugs to enhance delivery efficiency and therapeutic outcomes in bone
   and joint disorders [[47][17], [48][18], [49][19]]. In this study, the
   physical and chemical properties of the nanogels, including particle
   size and stability, were characterized to ensure their suitability for
   siRNA delivery. In vitro biocompatibility assessments with BMSCs
   confirmed the nanogels' safety and effectiveness in cellular
   applications. Comprehensive analysis demonstrated the nanogels' ability
   to silence genes in macrophages, inducing a phenotype shift towards the
   M2 pro-healing state. This study marks the use of TA-siRNA nanogels for
   targeted gene delivery to improve bone fracture healing, providing a
   new strategy for the development of biomimetic materials for complex
   bone regeneration in regenerative medicine.

2. Methods and materials

2.1. Data acquisition, standardization, and analysis of gene expression
Omnibus (GEO) data sets

   Data sets relevant to bone fracture healing were systematically
   searched and selected from the GEO database. Specifically, two GEO
   datasets, [50]GSE161315 and [51]GSE53256, were chosen based on their
   relevance to the study. [52]GSE161315 contains transcriptomic data
   comparing normal healing with delayed healing in Rattus norvegicus,
   while [53]GSE53256 includes data comparing young and old rats. The raw
   data from both datasets were downloaded for further analysis.

   The raw data were preprocessed and normalized using the Robust
   Multi-array Average (RMA) method to correct for any technical
   variations and to ensure consistency and comparability across samples.
   Box plots were generated to visualize the distribution of gene
   expression values across the samples and to identify any outliers.

   Principal Component Analysis (PCA) was performed to reduce the
   dimensionality of the data and to identify potential clustering based
   on different experimental conditions. The PCA plots were generated to
   show the separation of samples into distinct groups: normal vs. delayed
   healing for [54]GSE161315 and young vs. old for [55]GSE53256.
   Differential expression analysis was conducted using the "limma" R
   package to identify genes with significant changes in expression
   between the different groups. The analysis provided a list of
   differentially expressed genes (DEGs) with corresponding statistical
   significance values. Volcano plots were used to visualize the
   upregulated and downregulated genes in each dataset.

   Heatmaps were generated to visualize the expression patterns of the
   DEGs across the samples. The heatmaps illustrated distinct clustering
   of genes and samples, further supporting the results from the
   differential expression analysis. To understand the biological
   significance of the DEGs, Gene Ontology (GO) and Kyoto Encyclopedia of
   Genes and Genomes (KEGG) pathway enrichment analyses were performed. A
   Venn diagram was created to show the overlap of DEGs between the two
   datasets, indicating common and unique genes involved in the healing
   process. GO enrichment analysis revealed significant biological
   processes, while KEGG pathway analysis identified key signaling
   pathways involved in bone healing.

   A Protein-Protein Interaction (PPI) network was constructed to identify
   key genes based on their connectivity and functional significance.
   Several central genes, including Ccl4, were highlighted as significant
   nodes within the network, as identified by different centrality
   measures such as MCC, DMNC, and MCODE clustering.

2.2. Isolation and characterization of BMSCs

   BMSCs were harvested from the medullary cavities of both humeri and
   femurs of 2-week-old female SD rats. The BMSCs were subsequently
   cultured in low-glucose Dulbecco's Modified Eagle Medium (DMEM),
   enriched with 10 % fetal bovine serum (FBS; Gibco), and maintained at a
   temperature of 37 °C in an atmosphere containing 5 % CO[2.]

2.3. Preparation of self-Assembled TA@SiCcl4 nanogels

   To begin, prepare a solution of siRNA at a concentration of 0.2 O.D/mL
   and a TA solution at 0.2 w/v% using nuclease-free PBS. Place 1 mL of
   the siRNA solution in an ice bath and stir at 100 rpm for 3 min using a
   magnetic stirrer. Then, add 1 mL of the 0.2 w/v% TA solution dropwise
   to the siRNA solution and react for 30 min. Transfer the mixture to a
   100 kDa centrifugal filter unit (Amicon Ultra-4 Ultracel-100 K,
   Millipore) and dialyze for 24 h to remove unreacted TA and siRNA.
   Afterwards, add 200 μL of nuclease-free PBS to the nanogel, and store
   the TA-siRNA nanogel stock solution at 4 °C.

2.4. Preparation of biomimetic nanovesicles MTA@SiCcl4

   Cultivate Raw cells in vitro in large quantities, scrape them from the
   culture dish with a cell scraper, and resuspend in enzyme-free PBS to
   obtain a cell suspension. Add the cell suspension to a hypotonic lysis
   buffer containing protease inhibitors. Place the solution in an ice
   bath for 15 min, then subject it to repeated freeze-thaw cycles in
   liquid nitrogen, and perform differential centrifugation to obtain the
   RAW cell membrane. Thoroughly mix the TA-siRNA nanogel solution with
   the cell membrane solution in a 1:1 mass ratio to produce MTA@SiCcl4.
   Sequentially extrude this solution through manual extruders with 400 nm
   and 200 nm porous polycarbonate membranes, respectively, to finally
   prepare the biomimetic nanovesicles MTA@SiCcl4. Store the collected
   gene-carrying biomimetic nanovesicles MTA@SiCcl4 in water at 4 °C for
   future use.

2.5. Characterization of self-Assembled TA-siRNA nanogels and biomimetic
nanovesicles MTA@SiCcl4

   The successful synthesis of TA-siRNA nanogels is confirmed by the
   Tyndall effect. To do this, prepare TA, siRNA, and TA-siRNA solutions
   and illuminate with infrared light; the appearance of the Tyndall
   effect indicates successful synthesis of the TA-siRNA nanogels. Examine
   the morphology of the nanogels using Transmission Electron Microscopy
   (TEM): drop the prepared solutions on a 200-mesh copper grid, allow to
   sit at room temperature for over 6 h to ensure evaporation of water,
   and observe with TEM. Measure the size distribution and zeta potential
   of the nanogels using a dynamic light scattering analyzer (Zetasizer
   Nano ZS, Malvern). Phagocytosis experiment: Seed RAW cells into a
   six-well plate, add medium, and culture for 6 h to allow cells to
   adhere. Under sterile conditions, prepare the original TA-siRNA nanogel
   solution and biomimetic nanovesicles MTA@siCcl4, labeling the TA-siRNA
   nanogels with FITC (green light) and Raw cell membranes with Dil
   (orange-red dye). Add TA-siRNA or MTA@SiCcl4 to the well plate,
   incubate in a culture incubator for 2 h, then observe cell phagocytosis
   with confocal laser microscopy.

2.6. In vitro stability and Bioactivity Testing

2.6.1. In vitro cytocompatibility analysis

   In order to conduct in vitro cytocompatibility examination, the
   live/dead staining, proliferation test, and β-actin Staining Kit
   (Abkine) were employed to evaluate each sample. The survival of
   5 × 10^5 RAW cells were evaluated using calcein-AM/ethidium
   (CalceinAM/PI, Invitrogen). Each sample was cultured on a 12-well plate
   one day later. The BMSCs were incubated for 20 min at 37 °C after being
   combined with a culture mixture containing 3 M Calcein-AM and 5 M PI.
   The quantification of RAW cells proliferation was conducted subsequent
   to their cultivation for durations of 1, 3, and 7 days in a 12-well
   plate supplemented with CCK-8 solution. A 100 μL/mL CCK-8 solution was
   added to 12-well coculture plates containing RAW cells and samples. The
   supernatant's optical density (OD) values were determined at 450 nm
   using a microplate analyzer (BioTech) after 2 h. The cytoskeleton of
   2.5 × 10^5 BMSCs was examined by staining them with Actin-Tracker Green
   and Hoechst after a 3-day culture period in a 12-well plate.
   Microscopic images were acquired utilizing a confocal laser scanning
   microscope manufactured by Nikon.

2.6.2. The impact of Osteoimmunomodulation on osteogenic differentiation

   For co-cultured assays, RAW cells were plated in the upper chamber of
   Transwell, while the lower chamber contained the BMSCs. A polycarbonate
   membrane with 1.0 mm pores was used to separate the two cell types,
   facilitating cytokine exchange between them. 2.6.3. Alkaline
   Phosphatase (ALP) Staining and Quantification. The ALP staining kit
   (Beyotime) was used to stain BMSCs. After being fixed in 4 %
   paraformaldehyde for 30 min, the samples were stained with ALP dye
   according to the manufacturer's instructions. Following a duration of
   1 h, every sample underwent three rounds of washing with distilled
   water to prevent discoloration. Subsequently, the outcomes were
   examined and captured utilizing an optical microscope manufactured by
   Olympus. An ALP Assay Kit (Beyotime) was employed to quantify BMSCs.
   Following a period of 7 days dedicated to the induction of
   differentiation, the medium was then disposed of, and the plate
   underwent a gentle rinsing process with PBS three times. Subsequently,
   the alkaline phosphatase activity of BMSCs was assessed in accordance
   with the guidelines provided by the ALP quantitative analysis kit. The
   ALP activity at a wavelength of 450 nm was evaluated using a microplate
   analyzer manufactured by BioTech.

2.6.3. The process of gene expression

   The extraction of total RNA was performed using Trizol. Afterwards, it
   was converted into complementary DNA (cDNA) through reverse
   transcription using a reverse transcription kit (Takara). The
   experiment was carried out with the LightCycler 480II and the
   LightCycler 480 SYBR Green I Master apparatus manufactured by Roche.
   The experimental procedure was conducted in triplicate, and the
   quantitative analysis was performed using the 2^−ΔΔCt method.

2.6.4. Immunofluorescence and western bolt

   Following a 30-min period of paraformaldehyde fixation, the cells were
   treated with PBS supplemented with 0.1 % TritonX-100 and 3 % BSA at
   ambient temperature. After a duration of 1 h, the suitable primary
   antibodies were introduced and left to incubate overnight at a
   temperature of 4 °C. Subsequently, the secondary antibodies were
   injected for a duration of 1 h. Micrographs were captured using a
   confocal reflection microscope (Leica) subsequent to the application of
   Hoechst dye to the nuclei (Sigma). The Western blot analysis was
   conducted. The cells were lysed on ice using the CWBIO lysis solution,
   which contained protease and phosphatase inhibitors (Thermo Fisher).
   The determination of protein concentrations was conducted utilizing a
   BCA protein assay kit. A protein slurry weighing 40 g was subjected to
   denaturation at a temperature of 100 °C for a duration of 10 min. The
   proteins were then separated using SDS-PAGE electrophoresis and
   subsequently deposited onto polyvinylidene fluoride membranes (PVDF,
   Thermo Fisher). Following a 1-h period of immersion in 5 % skimmed
   milk, the membranes were subjected to overnight incubation with the
   primary antibodies at a temperature of 4 °C. Immunoblots were seen
   using an enhanced chemiluminescence (ECL) kit after 1 h of incubation
   with secondary antibodies (CST). The protein density was quantified
   using the ImageJ program.

3. Results

3.1. Data Retrieval, extraction, and analysis of GEO data sets

   The raw data for [56]GSE161315 and [57]GSE53256 were successfully
   downloaded and normalized using the RMA method. Box plots ([58]Fig. 1A)
   showed consistent distribution of gene expression values across the
   samples, indicating effective standardization. PCA plots ([59]Fig. 1B)
   revealed clear separation between normal and delayed healing groups in
   [60]GSE161315 and between young and old groups in [61]GSE53256,
   indicating distinct gene expression profiles. The differential
   expression analysis identified 142 DEGs in [62]GSE161315 (85
   upregulated and 57 downregulated) and 168 DEGs in [63]GSE53256 (96
   upregulated and 72 downregulated). Volcano plots ([64]Fig. 1C)
   illustrated these changes in gene expression.

Fig. 1.

   [65]Fig. 1
   [66]Open in a new tab

   Comprehensive Gene Expression and Interaction Analysis Post-Batch
   Effect Correction. (A) Boxplot illustrating the gene expression data
   after batch effect correction from datasets [67]GSE161315 and
   [68]GSE53256. (B) Principal Component Analysis (PCA) scatter plots
   showing the distribution of individuals and the separation between
   groups post-correction; ‘normal’ and ‘delayed’ in one, and ‘young’ and
   ‘old’ in the other, indicating variability and clustering within the
   datasets. (C) Volcano plots representing differential analysis between
   the two datasets, highlighting genes with significant expression
   changes: up-regulated (red), down-regulated (blue), and non-significant
   (gray). (D) Heatmaps displaying the expression patterns of
   differentially expressed genes across the two datasets, revealing
   distinct gene expression profiles between the compared groups.

   Heatmaps ([69]Fig. 1D) demonstrated distinct clustering of DEGs, with
   clear differentiation between the experimental groups, supporting the
   validity of the differential expression results. The Venn diagram
   ([70]Fig. 2A) showed 63 overlapping DEGs between the two datasets. GO
   enrichment analysis ([71]Fig. 2B) highlighted processes such as
   cytokine-mediated signaling and cell chemotaxis. KEGG pathway analysis
   ([72]Fig. 2C) identified pathways like cytokine-cytokine receptor
   interaction and NF-kappa B signaling, which are critical for bone
   healing. The PPI network analysis identified several key genes, with
   Ccl4 being prominently featured across different centrality measures
   ([73]Fig. 2D, E, and 2F).

Fig. 2.

   [74]Fig. 2
   [75]Open in a new tab

   Pathway Enrichment and Protein-Protein Interaction Network Analysis.
   (A) Venn diagram showing the overlap of differentially expressed genes
   between datasets [76]GSE161315 and [77]GSE53256. (B) GO enrichment
   analysis bubble chart, visualizing enriched biological processes with a
   color gradient representing p-value significance and bubble size
   denoting gene count. (C) KEGG pathway enrichment analysis bubble chart,
   displaying enriched pathways with the same visualization strategy as GO
   analysis. (D) PPI network generated using the Local Association
   Centrality (LAC) algorithm in CytoNCA, emphasizing the
   interconnectedness among the intersection genes. (E) The
   highest-scoring cluster module identified from the PPI network of all
   intersection genes using the MCODE algorithm, highlighting key gene
   interactions. (F) The top 10 pivotal genes ascertained by the MCC and
   DMNC topological analysis algorithms, indicating potential core genes
   in the network.

   This underscores the importance of Ccl4 in the regulatory network,
   suggesting its potential role in enhancing macrophage polarization and
   promoting bone healing. By integrating PCA, differential expression,
   enrichment analyses, and PPI network construction, we identified Ccl4
   as a crucial gene involved in the bone healing process, providing a
   foundation for potential therapeutic strategies targeting macrophage
   polarization.

3.2. Material characterization

   As shown in [78]Fig. 3A and B, neither the TA solution nor the siCcl4
   solution exhibits the Tyndall effect, which can be attributed to the
   uniform dispersion of molecules within the solutions. In contrast, the
   TA@siCcl4 solution displays the Tyndall phenomenon due to the
   self-assembly of TA and siCcl4 into nanoparticles, which scatter light
   and thus cause this effect. [79]Fig. 3C indicates that the TA-siRNA
   nanogels range in size from 100 to 150 nm, and compared to the
   TA@siCcl4 nanogels, the cell membrane-wrapped MTA@siCcl4 exhibits a
   distinct core-shell structure. Furthermore, the laser confocal
   colocalization image in [80]Fig. 3D confirms the successful preparation
   of the biomimetic nanovesicles MTA@siCcl4. Electrophoretic light
   scattering assays show that the zeta potentials of TA@SiCcl4 and
   MTA@SiCcl4 nanogels are −34.02 ± 6.43 mV and −31.12 ± 3.12 mV,
   respectively ([81]Fig. 3E). These measurements indicate the colloidal
   stability of the nanogels, with both types displaying potentials that
   suggest moderate stability, although MTA@siCcl4 shows a slightly less
   negative potential. [82]Fig. 3F demonstrates that compared to
   TA@siCcl4, MTA@siCcl4 is more effectively engulfed by RAW264.7 cells
   within the same timeframe, indicating its superior cell-targeting
   capability. [83]Fig. 3G shows no significant change in the particle
   size of MTA@siCcl4 and TA@siCcl4.

Fig. 3.

   [84]Fig. 3
   [85]Open in a new tab

   Characteristics of TA-siRNA Nanocomplexes and Biomimetic Nanovesicles
   MTA@SiCcl4. (A) Schematic diagram of synthesis of TA-siRNA nanogel. (B)
   The Tyndall effect confirms the synthesis of TA-siRNA, indicated by the
   light scattering in TA-siRNA solutions, unlike the homogeneous TA and
   siRNA solutions. (C) Size range and core-shell morphology of the
   TA-siRNA and MTA@SiCcl4 are visualized via TEM, with a notable
   core-shell structure evident for the biomimetic vesicles (Scale
   bar = 100 nm). (D) The successful formulation of biomimetic
   nanovesicles MTA@SiCcl4 is corroborated by confocal co-localization
   imaging, denoting proper assembly (Scale bar = 1 μm). (E) Particle size
   analysis shows an average zeta potential of −35.32 ± 8.32 for TA@SiCcl4
   and -46.32 ± 9.16 for MTA@SiCcl4. (F) Phagocytosis assay results
   highlight enhanced uptake of MTA@SiCcl4 by Raw 264.7 cells over
   TA@SiCcl4, suggesting superior cell-targeting capability (Scale
   bar = 20 μm). (G) Stability tests reveal no significant change in
   particle size for both MTA@SiCcl4 and TA@SiCcl4, indicating robust
   stability. Figures presented as Portable Network Graphics (PNG) with
   true color at 24 bits per pixel and dimensions of 1096x986 pixels,
   interlaced for optimal web display.

   These findings collectively contribute to the understanding of the
   physicochemical properties and cellular interactions of the synthesized
   nanogels, which are crucial for their potential applications in drug
   delivery and biomedical research.

3.3. In vitro biocompatibility of MTA@SiCcl4 nanocomplexes

   The evaluation of biocompatibility for novel biomaterials is crucial
   for their application in regenerative medicine. This study extensively
   examined the interactions between TA-siRNA nanocomplexes and MTA@SiCcl4
   biomimetic nanovesicles with RAW264.7 macrophages to assess their
   suitability. Live/dead staining indicated that macrophage viability
   exceeded 95 % across all experimental conditions, as corroborated by
   quantitative analysis ([86]Fig. 4A–C). These results suggest that the
   biomaterials are non-toxic and compatible with macrophages.
   Morphological analysis through actin staining demonstrated significant
   cellular adhesion and spreading on the surfaces of these biomaterials,
   indicating active cellular processes ([87]Fig. 4B). This was
   quantitatively confirmed by measuring the cell spread area,
   illustrating that the materials support cellular attachment and
   spreading ([88]Fig. 4D). Moreover, the CCK-8 assay revealed effective
   proliferation of BMSCs in the presence of these materials over a
   seven-day period, confirming that cellular growth and function remain
   intact ([89]Fig. 4E). The consistent results across all groups
   underscore the potential of these biomaterials for bone tissue
   engineering. Statistical analysis showed no significant differences
   among the groups, reinforcing the materials’ suitability for further in
   vivo studies.

Fig. 4.

   [90]Fig. 4
   [91]Open in a new tab

   Biocompatibility of each group. (A) Representative live/dead staining
   photos for RAW after contacting with siRNA, MTA@siCcl4 for 1 day. Red
   staining indicates dead cells, and green staining indicates live cells.
   Scale bar represents 200 μm. (B)The cytoskeleton images show the
   adhesion of RAW cultured on each sample after 3 days following culture.
   Scale bar represents 100 μm. (C)Quantitative analysis of live cells
   (n = 9). (D)Quantitative analysis of cell spread area (n = 9). (E)The
   CCK-8 assay was measured in each group after 1, 3, and 7 days of BMSCs
   culture (n = 5). ANOVA followed by Bonferroni's multiple comparison
   test was used for statistical analysis (∗p < 0.05, ∗∗p < 0.01,
   ∗∗∗p < 0.001).

   The findings support the potential integration of these nanocomplexes
   and vesicles in bone regeneration therapies, offering a compatible
   interface for cellular growth and function.

3.4. RAW264.7 cells Respond to inflammation

   MTA@SiCcl4 enhances macrophage phenotypic reprogramming in vitro. To
   determine the impact of MTA@SiCcl4 on macrophage polarization,
   RAW264.7 cells were incubated with PBS, siCcl4, TA@SiCcl4, and
   MTA@SiCcl4. The markers indicative of M1 (iNOS) and M2 (Arg-1)
   macrophage phenotypes were identified through immunofluorescence
   staining. [92]Fig. 5A illustrates a pronounced shift in RAW264.7 cells
   treated with MTA@SiCcl4 towards the M2 phenotype from the M1 state. The
   use of MTA@SiCcl4 treatment led to a notable 1.8-fold drop in iNOS
   expression, which indicates M1 polarization. Additionally, there was a
   rise in Arg-1 expression by roughly 2.3-fold, showing a reduction in
   the M2 pro-inflammatory condition. To delve deeper into the influence
   of MTA@SiCcl4 on macrophage polarization, the transcriptional levels of
   genes associated with M1 polarization, including iNOS, IL-6, and TNF-α,
   and those with M2 polarization, including IL-10 and Arg-1, were
   examined using reverse transcription RT-qPCR, as depicted in [93]Fig.
   5D and E. RT-qPCR confirmed these alterations, revealing a 55 %
   decrease in iNOS mRNA levels and a 120 % increase in Arg-1 mRNA levels.
   In comparison to the control group, the pro-inflammatory cytokines IL-6
   and TNF-alpha were substantially reduced by 55 % and 60 %,
   respectively, in the groups treated with MTA@SiCcl4, demonstrating the
   anti-inflammatory properties of the nanovesicles. In contrast, the
   levels of the anti-inflammatory cytokine IL-10 increased by 120 %,
   further supporting the transition towards a healing-promoting M2
   phenotype. Furthermore, there was a significant drop in the expression
   of the Ccl4 gene, with a 70 % reduction compared to the control group.
   This confirms that the siRNA-mediated silencing within the
   nanocomplexes was successful. This decrease verifies siRNA transport
   and functional activity and suggests that MTA@SiCcl4 nanovesicles may
   alter macrophage behavior toward a regenerative phenotype.

Fig. 5.

   [94]Fig. 5
   [95]Open in a new tab

   In vitro RAW264.7 cell polarization. (A) Arg-1 and iNOS(red)
   immunofluorescent staining of RAW264.7 cells on each group surface 3
   days after culture. Scale bar represents 50 μm. (B, C)) Quantitative
   analysis of the fluorescence intensity of iNOS and Arg-1 in each group
   (n = 3). (D, E) RT-qPCR was used to evaluate the effects of each group
   on the expression of inflammatory genes (n = 3). ANOVA followed by
   Bonferroni's multiple comparison test was used for statistical analysis
   (∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001).

   These findings demonstrate that MTA@SiCcl4 nanovesicles not only
   promote macrophage polarization towards an M2 phenotype but also
   effectively modulate the inflammatory gene expression profile in
   RAW264.7 cells, including the targeted silencing of Ccl4. This
   modification in the function of macrophages is essential for
   diminishing inflammation and creating a favorable environment for bone
   regeneration. The capacity to manipulate the phenotype of macrophages
   through targeted administration of siRNA demonstrates a promising
   approach to enhance the effectiveness of regenerative medicine in the
   treatment of complicated bone fractures.

3.5. Activation of M2-Polarized macrophages promote BMSCs differentiation

   BMSCs play a crucial role in bone regeneration, primarily through their
   differentiation into osteoblasts, essential for effective bone repair.
   The interaction between BMSCs and their microenvironment, especially
   with immune cells such as macrophages [[96]20], significantly
   influences BMSC fate. This study builds on recent insights that
   M2-polarized macrophages foster an osteogenic environment20, assessing
   the impact of M2-polarized RAW264.7 cells on the differentiation of
   BMSCs in a co-culture system. M2 macrophages were co-cultured with
   BMSCs under osteogenic conditions. Osteogenic differentiation was
   evaluated using several markers, including the enzymatic activity of
   ALP, and the expression of osteogenic markers such as RunX2 and
   osteopontin (OPN). Seven days post-co-culture, ALP activity was
   quantitatively analyzed, showing significantly elevated levels in BMSCs
   co-cultured with M2 macrophages compared to controls ([97]Fig. 6A and
   B). Immunofluorescent staining was conducted seven days
   post-co-culture, revealing substantial increases in RunX2 and OPN,
   indicative of enhanced osteoblastic differentiation ([98]Fig. 6C and
   D). Western blot analysis further corroborated these findings,
   indicating increased protein levels of RunX2 and OPN after seven days
   ([99]Fig. 6E and F). These findings were supported by RT-qPCR, which
   showed elevated mRNA levels for collagen type I, RunX2, and OPN,
   suggesting transcriptional regulation as a mechanism for enhanced
   differentiation ([100]Fig. 6G).

Fig. 6.

   [101]Fig. 6
   [102]Open in a new tab

   Macrophage polarization effect on BMSCs osteogenesis. (A)ALP staining
   of BMSCs cultivated in a coculture system for 7 days. Scale bar
   represents 200 μm (B) Quantitative analysis of ALP staining (n = 5).
   (C) BMSCs were immunofluorescently stained with RunX2 and OPN 7 days
   after being cocultured. Scale bar represents 50 μm. (D, E) Quantitative
   analysis of immunofluorescent staining (n = 3).(F) Western blot
   analysis was used to assess RunX2 and OPN protein expression after 7
   days. (G) Quantitative analysis of Western blot (n = 3). (H, I) After
   BMSCs were cocultured for 7 days (n = 3), RT-qPCR was used to evaluate
   theosteogenesis, including COL-1, RunX2 and OPN.ANOVA followed by
   Bonferroni's multiple comparison test was used for statistical analysis
   (∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001).

   The consistent results across different markers underscore the
   potential of M2-polarized macrophages to significantly enhance outcomes
   in bone tissue engineering. The observed upregulation of osteogenic
   markers provides compelling evidence of the therapeutic potential of
   M2-polarized macrophages in bone regeneration. This approach may pave
   new pathways for regenerative therapies that utilize the interplay
   between the immune system and stem cells for efficient and effective
   bone repair.

4. Discussion

   Our study explored the impact of MTA@SiCcl4 nanovesicles on macrophage
   polarization and subsequent bone regeneration, integrating extensive
   gene expression analyses, nanomaterial characterization, and detailed
   assessments of cellular responses. Material characterization confirmed
   the effective nanoparticle formation and stability of TA@siRSOP2 and
   MTA@siRSOP2 nanogels, with a consistent size range of 100–150 nm
   suitable for biological applications and a core-shell structure
   essential for their function as delivery vehicles. The colloidal
   stability, indicated by the Tyndall effect and appropriate zeta
   potential values, is critical for the biological functionality of these
   nanovesicles.

   In terms of biocompatibility and cellular response, the nanovesicles
   exhibited high viability in macrophage cultures, maintaining over 95 %
   cell viability and promoting cellular adhesion and proliferation. This
   underscores their potential for in vivo applications in tissue
   engineering. Moreover, the treatment with MTA@SiCcl4 nanovesicles
   resulted in a significant shift in macrophage behavior, reprogramming
   RAW264.7 cells from a pro-inflammatory M1 phenotype to a regenerative
   M2 phenotype. This transition was marked by notable changes in gene
   expression, including a decrease in pro-inflammatory markers and an
   increase in anti-inflammatory and regenerative markers.

   The enhanced osteogenic differentiation of BMSCs co-cultured with
   M2-polarized macrophages underscores the effectiveness of MTA@SiCcl4
   nanovesicles in fostering a microenvironment conducive to bone
   regeneration. The upregulation of critical osteogenic markers such as
   ALP, RunX2, and osteopontin, following co-culture with M2 macrophages,
   indicates a significant enhancement in bone matrix formation and
   mineralization, crucial for successful bone repair.

   Our research extends the current scientific frameworks by elucidating
   the complex interactions between siRNA-mediated interventions and
   macrophage functionality within the bone regeneration context. By
   leveraging MTA@SiCcl4 nanovesicles to modify macrophage behavior, we
   elucidate a vital mechanism wherein localized gene silencing
   significantly impacts tissue repair and regeneration outcomes. Notably,
   siRNA targeting the Ccl4 gene markedly reduces its expression in
   macrophages, facilitating a shift from a pro-inflammatory (M1) to a
   regenerative (M2) phenotype. This shift is critical as M2 macrophages
   play a key role in reducing inflammation and promoting healing,
   aligning with a growing body of evidence that supports macrophage
   phenotype manipulation as a therapeutic strategy to enhance
   regenerative processes [[103]21].

   Furthermore, the therapeutic potential of siRNA in bone regeneration is
   affirmed by its capability to precisely adjust the molecular
   environment at the bone repair site, promoting an anabolic state
   favorable for bone growth and remodeling. Our findings are consistent
   with recent research highlighting siRNA as a powerful and adaptable
   tool for manipulating key pathways in osteogenesis and bone homeostasis
   [[104]22]. These insights are crucial, as they validate the use of
   siRNA for immune modulation and pave the way for novel RNA
   interference-based therapeutic strategies tailored for complex
   orthopedic conditions [[105]23].

   Effective bone regeneration requires complex interactions among various
   cell types, including osteoblasts, osteoclasts, and macrophages.
   Macrophages, in particular, play a pivotal role in this process due to
   their ability to adopt different phenotypes that influence healing
   stages. Traditionally, macrophages transition from a pro-inflammatory
   M1 phenotype, which aids in clearing debris and pathogens, to an
   anti-inflammatory and pro-healing M2 state that supports bone repair
   [[106]24]. This phenotypic flexibility allows macrophages to adapt
   rapidly to environmental changes. In this study, we present initial
   evidence suggesting that silencing the Ccl4 gene promotes M2
   polarization, potentially enhancing the osteogenic process. Our data
   preliminarily suggest that Ccl4 silencing might facilitate a shift
   towards an M2 phenotype, which is known to secrete cytokines and growth
   factors that stimulate osteoblast activity and matrix formation
   [[107]11].

   The results of our study reveal a new feature of CCL4's function in
   macrophage biology. Specifically, we show that suppressing CCL4 leads
   to the promotion of M2 macrophage polarization, which in turn enhances
   bone regeneration. This observation contradicts previously established
   functions of CCL4, specifically its role in promoting the movement of
   preosteoclasts and its contribution to the formation of osteoclasts, as
   proposed by Lee et al. [[108]25] in their study on the interactions
   between CCL4 and CCR5.The disparity between our findings and prior
   research could be ascribed to the intricate nature of CCL4's
   involvement in immune modulation. Although Lee et al. [[109]25]
   highlighted CCL4's capacity to enhance preosteoclast cell migration via
   its receptor CCR5, contributing to osteoclast differentiation and bone
   resorption, our study indicates that in a different context, where
   macrophages are targeted for gene silencing, CCL4 can promote a
   reparative, anti-inflammatory macrophage phenotype. This shift in
   function underscores the versatile roles that immune mediators can play
   in different biological contexts.

   Moreover, the modulation of CCL4 expression might provide a valuable
   therapeutic tool to balance bone resorption and formation, particularly
   in diseases characterized by excessive inflammatory responses or bone
   degeneration. The use of CCL4 silencing to promote M2 macrophage
   polarization offers a promising avenue for enhancing bone healing,
   suggesting that manipulating CCL4 could mitigate conditions such as
   osteoporosis and rheumatoid arthritis by promoting bone regeneration
   and reducing inflammation.

   To create biomimetic nanovesicles MTA@SiCcl4, we next covered the outer
   layer of the nanogels with RAW264.7 cell membranes. The purpose of this
   process is to preserve the medicine from degradation and enable
   targeted drug administration. TA is a naturally occurring polyphenol
   that is derived from gallic acid and has amphiphilic properties. The
   structure of this substance includes several galloyl groups, which have
   the ability to interact with a diverse array of organic, inorganic,
   hydrophilic, and hydrophobic materials through hydrogen bonding,
   electrostatic interactions, coordination bonds, and hydrophobic
   interactions. Tannic acid, known for its natural crosslinking
   properties and its ability to reduce inflammation, fight bacteria, and
   inhibit cancer growth, has been extensively researched for its
   potential uses in the field of biomedicine [[110]16].

   The material's surface consists of vesicles originating from Raw cell
   membranes, employing a homologous targeting strategy to accomplish
   precise drug delivery to osteoclasts. Homologous targeting exploits the
   distinctive characteristics of biomimetic vesicles, which are designed
   to imitate the cell membranes of particular cell types, such as RAW
   cells in the context of bone-related treatments. These vesicles have
   the ability to selectively target and adhere to cells that possess
   comparable traits, therefore enabling the direct delivery of
   therapeutic medicines to the precise location of injury or disease.
   Studies have demonstrated that this focused strategy enhances the
   effectiveness of treatment and improves the results in many
   experimental models of bone and joint disorders [[111]17,[112]18]. The
   objective of this study is to overcome the drawbacks of existing bone
   fracture treatments by utilizing the distinctive characteristics of
   TA-siRNA nanogels and homologous targeting mechanisms. This has the
   potential to benefit patients with fractures caused by osteoporosis due
   to various reasons (such as medication factors) [[113]26].

5. Conclusion

   In conclusion, our study not only advances our understanding of the
   role of Ccl4 in bone healing but also introduces a novel therapeutic
   modality using MTA@SiCcl4 nanovesicles for targeted gene silencing. The
   potential to modulate immune responses and enhance regenerative
   outcomes through such targeted interventions opens new avenues for the
   development of advanced therapeutic strategies in bone fracture healing
   and other regenerative medicine applications.

CRediT authorship contribution statement

   Xianwen Ma: Writing – original draft, Methodology, Investigation,
   Conceptualization. Qi Zhou: Writing – review & editing, Visualization,
   Validation, Formal analysis, Data curation. Zhaofeng Liu: Writing –
   review & editing, Supervision, Software, Resources. Yibei Wang: Writing
   – review & editing, Supervision, Conceptualization. Yong Hu: Writing –
   review & editing, Project administration, Funding acquisition.

Declaration of generative AI and AI-assisted technologies in the writing
process

   During the preparation of this work the authors used Claude in order to
   improve readability and language. After using this tool, the authors
   reviewed and edited the content as needed and take full responsibility
   for the content of the publication.

Declaration of competing interest

   The authors declare the following financial interests/personal
   relationships which may be considered as potential competing interests:
   Yong Hu reports financial support was provided by Anhui Provincial
   Institute of Translational Medicine. Yong Hu reports financial support
   was provided by Anhui Medical University. If there are other authors,
   they declare that they have no known competing financial interests or
   personal relationships that could have appeared to influence the work
   reported in this paper.

Acknowledgement