Sequentially regulated immuno-osteogenic bioceramics enhance the regeneration of osteoporotic bone defects

Abstract

Sequentially regulating immune-osteogenesis to orderly transition from the inflammatory phase to bone reconstruction, and enhancing bone regeneration while inhibiting osteoclastic resorption, are crucial for the treatment of osteoporotic bone defect. Herein, the sequential loading and controlled release of interleukin-4 (IL-4) and alendronate (AL) based on the 3D-printed β-tricalcium phosphate (β-TCP) scaffolds were achieved via the Michael addition reaction combined with physical adsorption. IL-4 was rapidly released within two days, promoting M2 polarization of macrophage and secretion of anti-inflammatory and osteogenesis-associated factors. Meanwhile, AL was sequentially released over a two-week period, promoting the osteogenic differentiation of mouse bone mesenchymal stem cells (mBMSCs) while inhibiting the formation of osteoclasts. Bone regeneration efficacy in vivo was assessed using an osteoporotic rat femoral defect model. Microcomputed tomography (micro-CT) and histological analyses revealed that the functionalized scaffolds (TCP-PDA/AL-IL-4) exhibited superior osteogenic properties. Significantly, the bone mineral apposition rate (MAR) and the amount of newly formed bone increased in the TCP-PDA/AL-IL-4 groups, while the osteoclast population reduced. This study proposes a highly effective strategy for developing bone regeneration and repair materials specifically tailored for the osteoporotic bone defect.

Graphical abstract

1. Introduction

Bone repair is a dynamic and complex process that encompasses a series of biological events, including an initial pronounced inflammatory phase followed by a prolonged bone remodeling process [1,2]. Patients with osteoporosis exhibit an extended inflammatory phase and delayed excessive bone resorption due to abnormalities in the osteoimmune microenvironment [[3], [4], [5]]. Consequently, the regenerative repair process faces significant challenges when bone defects occur. The significant role of immune regulation in bone repair has been increasingly confirmed by numerous studies [[6], [7], [8]]. Macrophages are versatile immune cells that can dynamically modulate the osteoimmune microenvironment [[9], [10], [11]]. M1 macrophages secrete proinflammatory cytokines, thereby inducing inflammatory responses. Prolonged inflammation frequently leads to the development of a fibrous capsule around implant materials, hindering bone tissue regeneration. In contrast, M2 macrophages display an anti-inflammatory phenotype, facilitating tissue repair by secreting factors, such as the anti-inflammatory cytokine interleukin-10 (IL-10) transforming growth factor-β (TGF-β), and bone morphogenetic protein-2 (BMP-2) [[12], [13], [14]]. Therefore, timely regulating macrophage polarization toward the M2 phenotype modulates the osteoimmune microenvironment and reduces local tissue inflammation [15,16].

With the mitigation of the inflammatory response, bone repair progresses to the subsequent stage of bone reconstruction [17]. However, in an osteoporotic environment, the increased secretion of RANKL by osteoclast precursors and its interaction with receptor activator for nuclear factor-κB (RANK) promote osteoclast differentiation. This results in the production of hyperactive osteoclasts, increasing bone resorption beyond bone formation, thus hindering new bone development [18,19]. Enhancing osteogenic differentiation and inhibiting osteoclast formation are crucial during the bone reconstruction phase for the effective regeneration of osteoporotic bone defects [20,21]. Therefore, it is essential to design materials that can modulate the inflammatory microenvironment and promote bone regeneration, accelerating the repair process of osteoporotic bone defects.

Interleukin-4 (IL-4), a classical activator for inducing macrophage polarization toward the M2 phenotype [22], and alendronate (AL), a clinically proven treatment drug for osteoporosis [[23], [24], [25]] were chosen to address this issue. Functionalized scaffolds were constructed to sequentially modulate the initial immune response and subsequent bone reconstruction process by controlling the release of IL-4 and AL. As shown in Fig. 1, the early release of IL-4 polarizes macrophages into the M2 phenotype, mitigating the inflammatory response and indirectly promoting osteogenic differentiation by secreting various cytokines, establishing a conducive osteoimmune microenvironment. Subsequently, the continuous release of AL promotes bone formation and inhibits bone resorption, achieving sequentially regulating immune function and osteogenesis.

Fig. 1.

Schematic diagram of the mechanism of the functionalized scaffolds achieving sequential regulation of the bone repair process in osteoporotic conditions.

2. Materials and methods

2.1. Fabrication and characterization of samples

β-tricalcium phosphate (β-TCP) powder and scaffolds were prepared based on the method described on our previous research work [26]. β-TCP scaffolds were fabricated with the size of Φ10 × 3 mm for surface characterization and in vitro cell experiments [27,28]. Cylindrical scaffolds with the size of Φ 3 × 3 mm were prepared for in vivo femoral implantation. The sample preparation is illustrated in Fig. 2a. Dopamine hydrochloride was dissolved in a Tris-HCl buffer with a pH of 8.5 to obtain a 0.5 mg/mL solution. The β-TCP scaffolds were then immersed in this solution for 24 h at 37 °C under light-protected conditions to prepare TCP-PDA scaffolds. Subsequently, the PDA-modified scaffolds were transferred to an AL (Aladdin, USA) solution (2 mg/mL) for 24 h at 37 °C under light-protected conditions to obtain TCP-PDA/AL scaffolds, which were then dried for further use. Finally, 20 μL of IL-4 (PeproTech, USA) was added to the materials and dried overnight in a cell incubator to produce the TCP-PDA/AL-IL-4 scaffold with sequential regulation capabilities.

Fig. 2.

Sample preparation and analysis of physicochemical properties: (a) diagrammatic representation of the sample preparation procedure. (b) SEM images of different scaffolds. (c) XPS spectrum. (d–e) High-resolution C1s spectra of (d) TCP-PDA and (e) TCP-PDA/AL. (f) In vitro release profiles of AL and IL-4. (g) Surface contact angle measurements of different scaffolds. (h) BSA adsorption analysis.

The surface morphology was visualized using a field-emission scanning electron microscope (FE-SEM, Merlin, Zeiss, Germany). The structural characteristics of the scaffolds were analyzed using a fully automated laser Raman microscope (DXR2xi, Thermo Fisher, USA). A contact angle system (DSA25, Kruss, Germany) was used to evaluate the hydrophilicity of samples. A BCA Protein Assay Kit (Beyotime, China) was used to quantify protein adsorption. The surface chemical composition was analyzed through X-ray photoelectron spectroscopy (XPS) with an Escalab Xi + instrument (Thermo Fisher Scientific, USA).

2.2. Release of IL-4 and AL from scaffolds

At 37 °C, TCP-PDA/AL-IL-4 were incubated in phosphate-buffered saline (PBS) solution under gentle agitation to analyze the release behavior. Samples were collected at the scheduled time and replenished with an equal amount of fresh PBS. The release of AL was quantified by a UV–visible spectrophotometer (UV2600, Shimadzu, Japan) based on a reported study [29]. The release of IL-4 was assessed using a Mouse IL-4 enzyme-linked immunosorbent assay (ELISA) kit (Meimian, China). Three Samples were used and each Sample is independently repeated three times.

2.3. Cytocompatibility and cell proliferation

Mouse-derived macrophage RAW264.7 and mouse bone marrow mesenchymal stem cells (mBMSCs) were seeded on scaffolds with a density of 3 × 10⁴ cells per sample in 48-well plates. On days 1, 3, and 5, the medium was carefully removed from the wells, and the samples were washed with PBS. The blood compatibility of the scaffolds was evaluated using hemolysis test according to the ISO 10993–4:2017. Cell proliferation was assessed using the Cell Counting Kit-8 (CCK-8, Beyotime, China). The viability of the seeded cells on scaffolds for 3 days was evaluated using a live/dead cell assay (Beyotime, China). An Eclipse Ti-U inverted fluorescence microscope (Nikon, Japan) was used to observe the stained cells. 5 parallel samples were used for each experimental group.

2.4. Effects of samples on macrophage polarization behavior

2.4.1. Enzyme-linked immunosorbent assay (ELISA)

The medium was collected following a 3-day coculture of RAW264.7 cells with scaffolds. The levels of IL-10, tumor necrosis factor-α (TNF-α), and BMP-2 were quantified using an ELISA kit (Meimian, China). The concentrations of these factors secreted by RAW264.7 cells were determined based on the standard curve. Additionally, markers of the macrophage phenotype CCR7 and CD206 expression were also characterized. 5 parallel samples were used for each experimental group.

2.4.2. Immunofluorescence

After coculturing with scaffolds for 3 days, RAW264.7 cells were fixed with 4 % paraformaldehyde (Biosharp, China) on ice for 30 min. Following fixation, cells were permeabilized with Triton X-100 (Beyotime, China) and subsequently blocked with bovine serum albumin (BSA, Beyotime, China). The samples were incubated with primary antibodies at 4 °C overnight, and then stained in DAPI solution for 15 min at room temperature. A laser confocal microscope (TCS SP8, Leica, Germany) was used for imaging. 3 parallel samples were used for each experimental group.

2.4.3. Inflammation-related gene expression

RAW264.7 cells were seeded at a density of 5 × 10⁴ cells per sample on the scaffolds and cultured for 3 days. The expression levels of genes related to immune response and osteogenesis were evaluated through real-time polymerase chain reaction (RT-PCR). The expression of genes associated with inflammation, including Interleukin-6 (IL-6), chemokine receptor type 7 (CCR7) and TNF-α, Arginase-1 (Arg-1), IL-10 and Macrophage mannose receptor (CD206), and angiogenesis-related genes platelet-derived growth factor BB (PDGF-BB) and TGF-β were detected. Table S1 presents the forward and reverse primers. Gene expression was quantified using glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as the housekeeping gene, employing the 2^-△△CT calculation method. 5 parallel samples were used for each experimental group.

2.5. Osteogenic differentiation of BMSCs in macrophage-conditioned medium

2.5.1. Migration ability of BMSCs

The culture medium was collected after coculturing macrophages with different scaffolds for 3 days. After filtering and sterilizing with sterile filter membrane, the culture medium was then mixed with a specific medium of BMSCs to obtain a conditioned medium. mBMSCs cells were cultured with conditioned medium until the cells formed a cell monolayer, when DMEM high-glucose medium without serum was taken as a substitute, and starvation treatment was performed for 12 h. A sterile tip was used to draw the lines. The conditioned medium was added after washing off detached cell fragments gently with a PBS solution. At 0, 6, 12 h, a microscopic was used to take photos, and ImageJ software was used to analyze the cell migration rate.

2.5.2. Ability of conditioned media to contribute to osteogenic differentiation

mBMSCs were inoculated in 6-well plates at a density of 5 × 10⁵/well and cultured for 24 h. The medium was changed to conditioned medium to culture for 3 and 7 days. After the cells were treated with lysate, the liquid was centrifuged and the supernatant was collected. A BCA Protein Assay Kit (Beyotime, China) and an alkaline phosphatase (ALP) Assay Kit (Beyotime, China) were used to test total protein and ALP activity. Cells were stained with an ALP Staining Kit (Beyotime, China) and photographed with a stereomicroscope (S9D, Leica, Germany). The genes expression of osteogenic-related genes, such as runt-related transcription factor 2 (Runx-2), collagen type 1 (COL-I), vascular endothelial growth factor (VEGF), ALP, osteopontin (OPN) and osteocalcin (OCN) was quantitatively evaluated using RT-PCR. 5 parallel samples were used for each experimental group.

2.6. Osteogenic properties of bioceramic scaffolds with sequence-modulated functions

The medium was removed after mBMSCs were seeded on the scaffolds for 24 h. Following fixation with 4 % paraformaldehyde and permeabilization with 0.1 % Triton X-100, the F-actin cytoskeleton was stained and incubated for 2 h at 37 °C in the dark. DAPI staining was then performed for 15 min. After staining the cytoskeleton, a TCS SP8 laser confocal microscope (Leica, Germany) was used to observe the cell adhesion morphology.

mBMSCs cells were cocultured with sterilized scaffolds in 48-well plates at a density of 5 × 10⁴/sample. After 7 and 14 days of osteogenic induction, the expression of osteogenic-related genes (ALP, COL-I, OPN, OCN) and ALP activity were evaluated as per the protocols described in Tection 2.6.2. 5 parallel samples were used for each experimental group.

2.7. Osteoclastic properties of bioceramic scaffolds with sequence-modulated functions

RAW264.7 cells were seeded on the scaffolds at a density of 5 × 10⁴/sample. After cell adhesion, the medium was changed to DMEM complete medium containing RANKL (50 ng/mL) for 6 days to induce the differentiation of RAW264.7 cells to osteoblasts. Osteoblasts were stained with using a tartrate-resistant acid phosphatase (TRAP) Staining Kit (Wako, Japan) and photographed under a stereomicroscope (S9D, Leica, Germany). Meanwhile, Beyotime TRAP Assay Kit was used to test TRAP activity. The expression of osteoclast-related genes, such as TRAP, RANK, matrix metalloprotein 9 (MMP9), nuclear factor of activated T cells cytoplasmic 1 (NFATC-1), cathepsin K (CTSK), was quantitatively analyzed using RT-PCR according to the method described in Tection 2.6.2. 5 parallel samples were used for each experimental group.

2.8. In vivo animal experiments

Twelve-week-old Sprague Dawley female rats weighing 200–250 g were purchased from Hunan Slake Jinda Animal Experiment Center for animal experiment implantation in vivo. At each time point, 4 rats for each experimental group. The implantation experiments and protocols were approved by the Ethics Committee of the Animal Center, South China University of Technology.

2.8.1. Establishment of rat model with osteoporosis and material implantation

Osteoporotic rat models were created through bilateral ovariectomy. The femur was analyzed through micro-CT 3 months after surgery to confirm the success of the osteoporosis model. Subsequently, 2.5 % pentobarbital sodium was injected in the intraperitoneal at a dosage of 45 mg/kg to fully anesthetized the rats. The surgical procedure is shown in Fig. S8A. A 2-cm-long wound was made at the distal femoral joint of the rat with a scalpel to expose the position of femoral ankle. A dental drill was used to make a Φ 3 × 3 mm bone defect. Scaffolds were implanted into the bone defect, and the muscle and skin were carefully sutured. The rats were randomly divided into three groups (TCP, TCP-PDA/AL, and TCP-PDA/AL-IL-4) according to the implanted scaffolds.

2.8.2. Polarization behavior of macrophages on materials in vivo

The rats were euthanized after 7 days postoperation. After embedding in paraffin and sectioning, the specimens were stained with immunofluorescence using iNOS (M1 maker, Sabbiotech, China) and CD206 (M2 maker, Sabbiotech, China) primary antibody as well as a corresponding secondary antibody to detect macrophage polarization around the implants. Photographs were taken under a fluorescence microscope. Meanwhile, immunohistochemical staining was performed using Arg-1 (M2 maker, Affinity, USA) primary and secondary antibodies to examine the expression of signature proteins of the M2 phenotype.

2.8.3. Bone regeneration after femoral implantation in osteoporotic rats

At 6 weeks after operation, 3D images were reconstructed using micro-CT (Zhongkekaishen, China) to evaluate new bone formation. Based on density difference the Mimics software was used to differentiate new bone and remaining material. Subsequently, the removed femur was decalcified and embedded in paraffin for hematoxylin and eosin (H&E) and TRAP staining. At 12 and 3 days before the execution of the rats, calcein (5 mg/kg) and dimethylphenol (25 mg/kg) were injected intramuscularly to evaluate the new bone formation and mineralization. After removing the femur, it was fixed in 4 % paraformaldehyde and dehydrated through a graded alcohol. The samples were embedded in polymethylmethacrylate and cut into 150-200-μm-thick slices along the sagittal direction of the central segment using a tissue slicer (SAT-001, Aolinjin, China) after hardening. The slices were then glued to a plastic holder and polished to 10–20 μm. Finally, fluorescent labeling of the new bone tissue was observed using a AiMco Austar43 fluorescence auto-scanning microscope. Quantitative processing was performed with the ImageJ software.

2.9. Statistical analysis

Data were presented as means ± standard deviation and analyzed using one-way analysis of variance, followed by a post hoc test. Statistical significance was set at p < 0.05.

3. Result and discussion

3.1. Characterization of the scaffolds

Fig. S1 shows the appearances of scaffolds. Scaffolds coated with PDA exhibited a darker coloration, while modifications using AL and IL-4 did not alter their surface appearance. As shown in Fig. 2b, the sintered β-TCP bioceramics exhibited well-defined grain boundaries and smooth surfaces. Following PDA modification, a black film containing numerous nanoparticles was formed on the scaffold surfaces. In contrast, there were no significant changes on the surfaces of TCP-PDA/AL and TCP-PDA/AL-IL-4 compared with that of TCP-PDA.

Polydopamine is a versatile, mussel-inspired material used in the formation of functional coatings. Polydopamine possesses abundant functional groups and exhibits secondary reactivity, enabling Michael addition and Schiff base reactions with biomolecules containing amino and sulfhydryl groups. The quinone functional groups (C=O) undergo ring-opening to participate in addition reactions with -NH2, forming C-O, C-N, and C=N bonds for further functional modification. The infrared spectral results are shown in Fig. S2b. A minor peak at 1615 cm⁻¹ was observed for both TCP-PDA and TCP-PDA/AL scaffolds, corresponding to the overlapping vibration of the C=C stretching and N–H bending in the indole structure of polydopamine. The Raman spectroscopy data shown in Fig. S2c indicate the success of PDA coating on the TCP scaffold surface. Distinct characteristic peaks were detected at 1350 cm⁻¹ and 1570 cm⁻¹, corresponding to the stretching and deformation vibrations of catechols, consistent with the findings of Ku et al. [30]. XPS analysis was performed to determine the chemical states of TCP-PDA and TCP-PDA/AL scaffolds. The high-resolution C1s spectra depicted in Fig. 2d and e reveal a low binding energy peak at 284 eV, which is attributed to the unsaturated bond in the aromatic ring of dopamine. The intensity of the C–O/C–N peak at 286.5 eV increased in the TCP-PDA/AL scaffold, while the C=O peak at 288.3 eV decreased, indicating that the C=O groups of dopamine successfully underwent Michael addition reactions with the −NH2 groups of AL. As shown in Fig. S2d and S2e, additional chemical changes can be observed from the high-resolution P2p and N1s spectra. Compared to that of the TCP-PDA scaffold, a phosphorus peak reappeared on the surface of TCP-PDA/AL scaffolds, due to the presence of phosphorus elements in AL. Moreover, the enhanced intensity of nitrogen peaks further confirmed the successful grafting of AL.

As shown in Fig. 2g, the average contact for TCP is 90°, which decreased to 45° following PDA coating. However, the introduction of AL and IL-4 did not significantly influence their hydrophilic properties. The PDA surface is enriched with hydrophilic functional groups, including amino and hydroxyl groups. These groups enhance the wettability of the material surface by facilitating the formation of hydrogen bonds, thereby improving its interfacial properties [31]. Compared with that of TCP groups, the protein adsorption in the other three groups was significantly enhanced. This finding is consistent with the trend observed in contact angle measurements, likely due to the nanostructure of PDA providing a greater number of adsorption sites. Materials with enhanced protein adsorption capacity are more conducive to recruiting various small molecules, mediating cell adhesion, and promoting tissue regeneration [32].

3.2. In vitro drug release of AL and IL-4

Results of sequential release of AL and IL-4 are shown in Fig. 2f. IL-4 was loaded onto the scaffold via physical adsorption, exhibiting an 80 % release within the initial 2 days, indicative of a burst release profile. In contrast, AL was covalently grafted onto the scaffold surface through a Michael addition reaction. Its release kinetics demonstrated an initial burst phase, accounting for 29 % release within the first 24 h, followed by a sustained and gradual release over the subsequent 14 days, finally achieving a cumulative release rate of 80 %. These findings are in agreement with the expected outcomes as illustrated in Fig. S3. 10⁻⁴ M, 10⁻⁵ M, 10⁻⁶ M and 10⁻⁷ M were chosen to identify the optimal loading concentration of AL based on the results from proliferation and differentiation experiments conducted with mBMSCs. The results are presented in Fig. S4. TCP-PDA/AL scaffolds loaded with a concentration of 10⁻⁵ M demonstrated the highest ALP expression, suggesting that this specific concentration of AL possesses the most potent capability to promote osteogenic differentiation. Therefore, a drug loading concentration of 10⁻⁵ M was chosen for subsequent studies.

3.3. In vitro immunomodulation

Modulating the initial immune response is essential for successful bone tissue regeneration [33,34]. The proliferation of RAW264.7 cells on different scaffolds is shown in Fig. 3a and b. RAW264.7 cells adhered to the scaffold surfaces in rounded clusters after 3 days of coculture, consistent with their typical growth behavior [35]. Notably, no red-stained dead cells were detected on any scaffold surface, indicating an absence of cytotoxicity across all groups. The OD values in the CCK-8 assay were significantly elevated after both 3 and 5 days of culture, accompanied by a marked increase in cell numbers. These results suggest that all tested scaffolds possess a robust capacity to promote RAW264.7 cell proliferation.

Fig. 3.

Research on macrophage polarization behavior: (a) Live/dead staining of cells cultured for 3 days. (b) Cell proliferation at 1, 3, and 5 days post coculture. (c–e) Secretion levels of TNF-α, IL-10, and BMP-2 following 3-day and 5-day coculture periods. (f) Expression of osteogenic-related genes following 3 days of culture.

To assess the polarization of macrophages, the secretion levels of relevant factors were quantified using an ELISA kit at 3 and 5 days post coculture, as shown in Fig. 3c and d. IL-10 is a recognized anti-inflammatory cytokine linked to M2-type macrophages, whereas TNF-α is a proinflammatory cytokine associated with M1-type macrophages. The TCP-PDA/AL-IL-4 scaffold exhibited significantly higher IL-10 secretion and markedly lower TNF-α secretion compared with the other three groups, indicating that effectively mitigates the inflammatory response and fosters a beneficial immune microenvironment during the early inflammatory phase. BMP-2 plays a crucial role in initiating osteoblast differentiation. The capacity of macrophages to stimulate bone formation through paracrine effects can be evaluated by assessing the expression levels of BMP-2 [36]. As shown in Fig. 3e, the TCP-PDA/AL-IL-4 group exhibited the highest BMP-2 expression among all groups at 3 and 5 days. Studies have demonstrated that TGF-β and PDGF-BB secreted by immune cells play significant roles in inducing mBMSC migration and promoting osteogenic differentiation [[37], [38], [39]]. As shown in Fig. 3f, the TCP-PDA/AL-IL-4 group was more effective in enhancing the expression of TGF-β and PDGF-BB.

The expression of macrophage surface markers was evaluated using immunofluorescence staining. The results are shown in Fig. 4a and S5. After a 3-day coculture, the TCP-PDA/AL-IL-4 scaffold exhibited the highest and lowest fluorescence intensities for CD206 protein staining and CCR7 protein staining, respectively. Genes associated with M1 macrophages included CCR7, TNF-α, and IL-6, while those related to M2 macrophages comprised CD206, IL-10, and Arg-1. Compared to the TCP group, the remaining three groups demonstrated significantly lower expression levels of M1-related genes, as shown in Fig. 4b–h. Notably, the TCP-PDA/AL-IL-4 scaffold showed the lowest expression of CCR7 and IL-6 genes. Conversely, gene expression associated with the M2 phenotype exhibited an opposing trend. The anti-inflammatory gene expression in the TCP-PDA/AL-IL-4 scaffold was significantly higher than that of the other three groups, indicating the effective suppression of the initial inflammatory response. These results can be attributed to the regulatory effect of IL-4 on macrophage polarization.

Fig. 4.

In vitro immunomodulation: (a) Immunofluorescence staining after 3 days of coculture (red: CCR7, green: CD206). (b–h) Gene expression related to inflammation following 3 days of culture (M2: IL-10, CD206, Arg-1; M1: IL-6, CCR7, TNF-α). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

3.4. Macrophage-conditioned medium enhances the osteogenic differentiation of mBMSCs

Macrophages act as a powerhouse and secrete a series of cytokines to mediate cell proliferation and migration, stimulate angiogenesis, and facilitate tissue remodeling [40,41]. We performed a scratch assay to investigate the effects of conditioned medium from the macrophage on the migration ability of mBMSCs. As illustrated from the results in Fig. S6, the scratched areas in all groups progressively diminished with increasing culture time. Notably, mBMSCs cultured in the TCP-PDA/AL-IL-4 group exhibited the most extensive migration at 12 h. Quantitative analysis using ImageJ software revealed that the TCP-PDA/AL-IL-4 group achieved a migration rate of 30 % at 12 h, which was 1.5–2 times higher than that of the other three groups. The result indicates that the conditioned medium from the TCP-PDA/AL-IL-4 group possesses the strongest migratory-promoting effect on mBMSCs.

To evaluate the influence of macrophage paracrine activity on osteogenic differentiation, mBMSCs were cultured in the conditioned media from each group for 4 and 7 days, as illustrated in Fig. 5a. ALP is a well-established marker to evaluate the extent of osteogenic differentiation [42]. The results are shown in Fig. 5b and c. Notably, the TCP-PDA/AL-IL-4 group showed the highest ALP expression and the most intense ALP staining at both 4 and 7 days, suggesting that the conditioned medium from this group was more effective in promoting osteogenic differentiation. To further verify the above finding, the expression levels of osteogenic-related genes in mBMSCs were assessed using RT-PCR. The results are shown in Fig. 5d–i. The highest expression of the early transcription factor Runx-2 was found in the TCP-PDA/AL-IL-4 group, indicating that this group exhibited the earliest initiation of the osteogenic differentiation process. The expression of OCN progressively increased with the prolongation of culture time, suggesting that mBMSCs in the TCP-PDA/AL-IL-4 group achieved the highest degree of osteogenic differentiation. As previously discussed, bioceramics with sequential regulatory functions effectively promoted M2 polarization of macrophages and secreted a range of osteogenic cytokines (including BMP-2, TGF-β, PDGF-BB), thereby enhancing osteogenic differentiation of mBMSCs. These results indicate that the TCP-PDA/AL-IL-4 group possesses optimal osteoimmunomodulation capacity and provides an excellent osteogenic immune microenvironment conducive to bone regeneration.

Fig. 5.

Osteogenic differentiation of mBMSCs in macrophage-conditioned medium: (a) Schematic diagram of conditioned medium preparation. (b–c) ALP staining and ALP activity after 4 and 7 days of culture. (d–i) Expression of osteogenic-related genes (Runx-2, ALP, COL-I, OPN, OCN, VEGF) after incubation for 4 and 7 days.

3.5. Regulation of osteoblastic and osteoclastic differentiation in vitro

Live/dead staining result (Fig. 6a), the proliferation result(Fig. 6c) and the hemolysis test result (Fig. S7) indicated that all tested scaffolds demonstrated satisfactory biocompatibility. Cell adhesion and spreading serve as the foundation for achieving other cellular functions. Cells that exhibit extensive spreading are more likely to differentiate into osteoblasts [43]. The adhesion and growth patterns of mBMSCs were examined after 24 h of culture on the scaffolds. As shown in Fig. 6b, cells on the surface of TCP scaffold surface displayed an elongated morphology, whereas those on the modified scaffold exhibited a flattened shape with a larger spreading area and increased pseudopod protrusions. This may be attributed to PDA nanoparticle coating that provided additional adhesion sites, thereby facilitating the mediation of biochemical signals and enhancing the ability of cells to sense their surroundings, which in turn regulated their biological behaviors. Fig. S8 shows the unit spreading area of mBMSCs, indicating that the average spreading area of cells in the TCP-PDA/AL-IL-4 group was approximately twice that of the TCP group.

Fig. 6.

Effects of each group of scaffolds on the osteoblast and osteoclast behavior: (a) Live/dead staining of mBMSCs culturing for 3 days. (b) Morphological characteristics of mBMSCs after 24 h of culture. (c) Cell proliferation of mBMSCs at 1, 3, and 5 days. (d–e) ALP activity following 7 and 14 days of incubation. (f–i) Expression levels of osteogenesis-related genes after 7 and 14 days of osteogenic induction. (j) TRAP activity of RAW264.7 cells. (k) Expression of osteoclast-related genes (NFATC-1, MMP9, CTSK, TRAP, RANK) of RAW264.7 cells.

The ALP activities of all modified scaffolds were significantly enhanced following 7 and 14 days of coculture, with the TCP-PDA/AL-IL-4 scaffold exhibiting the highest activity on day 14 (Fig. 6d and e.). Results of osteogenic differentiation of mBMSCs are shown in Fig. 6f–i. After a 7-day coculture, the TCP-PDA/AL-IL-4 scaffold demonstrated a significant increase in COL-I expression, facilitating the formation of fibro-soft tissues and promoting matrix remodeling. Additionally, a significant difference is found in the expression of OPN and OCN. By day 14, ALP, OPN, and OCN exhibited a substantial upregulation in the TCP-PDA/AL-IL-4 group. Overall, TCP-PDA/AL-IL-4 scaffolds exhibited the most favorable expression of osteogenic-related genes, attributed to the sustained release of AL, which positively influenced osteoblast differentiation.

Excessive osteoclast-mediated bone resorption is an important cause of osteoporosis [44]. Regulating osteoblast and osteoclast differentiation by inhibiting osteoclast activity is an effective approach to repair osteoporotic bone defect. TRAP activity serves as a biomarker for evaluating osteoclast function and can indicate the extent of osteoclast differentiation. Elevated TRAP activity correlates with increased osteoclast activity [45]. As shown from the results in Fig. 6j, the expression of TRAP was significantly reduced in modification scaffolds. Notably, the TCP-PDA/AL-IL-4 scaffolds exhibited the lowest level among all groups. At the same time point, the expression of genes related to osteoclast differentiation, such as TRAP, RANK, NFATC-1, MMP9, and CTSK was assessed. The results are shown in Fig. 6k. Collectively, these results demonstrate that TCP-PDA/AL-IL-4 groups markedly downregulate the expression of osteoclast-related genes, exerting the strongest inhibitory effect on osteoclast differentiation.

3.6. Bone regeneration in osteoporotic rats

Oophorectomy is a well-established method for modeling osteoporosis in animal studies [46]. The sagittal plane of the femur was examined and reconstructed in 3D using micro-CT (Fig. S11a). Compared with NBR rats, OVX rats showed a significant reduction in trabecular bone number, with sparse density and irregular arrangement. Further analysis of bone-related parameters (Fig. S11b–S11f) revealed that bone mineral density (BMD), trabecular number (Tb.N), bone volume fraction (BV/TV), and trabecular thickness (Tb.Th) were significantly lower in the OVX group, reaching only one-third to one-quarter of the values observed in the NRM group. Additionally, trabecular separation (Tb.Sp) was markedly increased, indicating a more sparse trabecular structure. These findings confirm that the osteoporosis model has been successfully established.

3.6.1. In vivo immunomodulation

Materials were implanted into the femoral defect for a period of 7 days to investigate their in vivo immunomodulatory effects. Subsequently, both the materials and the surrounding tissues were excised. The inflammatory status of the adjacent tissues was evaluated using immunofluorescence analysis. The results are shown in Fig. 7a, c, and 7d, demonstrating a significant difference in the impact of the scaffolds on macrophages across various groups. Compared with the TCP and TCP-PDA/AL groups, the TCP-PDA/AL-IL-4 group exhibited a significant increase in CD206-positive cells and enhanced positive expression (Fig. 7c). Conversely, there was a marked reduction in iNOS-positive cells and diminished iNOS expression (Fig. 7d). Immunohistochemical staining results are shown in Fig. 7b and e. Increased Arg-1-positive expression was found surrounding the TCP-PDA/AL and TCP-PDA/AL-IL-4 groups, with the TCP-PDA/AL-IL-4 group demonstrating the largest areas of positive staining and the most intense staining intensity. These results align with the results of in vitro cell experiments. The TCP-PDA/AL-IL-4 scaffolds, which possess sequential regulatory capabilities, effectively inhibit inflammation and significantly mitigate early inflammatory responses, thereby fostering a osteoimmune microenvironment that is more conducive to bone regeneration.

Fig. 7.

In vivo immunomodulatory effects of each group of scaffolds: (a) The results of CD206 and iNOS immunofluorescence staining after 7 days of in vivo implantation (red: positive cells). (b) Immunohistochemical staining of Arg-1 at day 7 postimplantation. (c–e) Quantitative analysis of the immunofluorescence and immunohistochemistry staining at day 7 postimplantation. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

3.6.2. Bone regeneration in vivo

The materials were implanted into femoral defects for 6 weeks to evaluate bone regeneration. The 3D reconstruction results are shown in Fig. 8a. The results showed that new bone formation occurred inwardly from the scaffold edges. Among all groups, the highest quantity of new bone tissue was observed within the TCP-PDA/AL-IL-4 scaffolds. The parameters analyzed for the new bone tissue included BV/TV, Tb.N, BS/TV, and Tb.Sp, with the results shown in Fig. 8b. Specifically, the BV/TV of the TCP-PDA/AL-IL-4 scaffolds was 27.04 % ± 1.3 %, significantly higher than the 20.52 % ± 2.4 % observed in the TCP group. Additionally, BV/TV in the TCP-PDA/AL-IL-4 scaffolds was significantly greater, while the dispersion of trabeculae was notably lower, indicating enhanced connectivity and improved morphology of the trabecular network. In summary, the TCP-PDA/AL-IL-4 scaffolds, which possess immune-osteogenic sequence regulation capabilities, exhibit superior bone regeneration promotion effects.

Fig. 8.

In vivo bone regeneration effect of each group after implantation for 6 weeks: (a) 3D reconstruction images (red: material; yellow: new bone, 120° section view). (b) Quantitative analysis of new bone: BV/TV, Tb.N, BS/TV, and Tb.Sp. (c) H&E staining after 6 weeks of materials implantation(M: material; NB: new bone). (d) Mineral apposition rate. (e) Double-labeling fluorescent image: fluorescence bands in the process of new bone formation (green: calcineurin; orange: dimethylphenol orange). (f) TRAP staining after 6 weeks of material implantation (black arrows mark TRAP-positive cells). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

H&E staining was conducted on tissue sections to further assess bone formation at the bone defect site, as shown in Fig. 8c. The TCP group exhibited a lower new bone formation, whereas the TCP-PDA/AL-IL-4 group demonstrated significantly greater new bone generation within the scaffolds. The trend in the quantity of new bone formation was as follows: TCP-PDA/AL-IL-4 > TCP-PDA/AL > TCP. Consistently, Masson staining results (Fig. S12) showed that the collagen fibers within the TCP-PDA/AL-IL-4 scaffolds were more orderly arranged, indicating a higher degree of maturity in the newly formed bone tissue. Furthermore, the bone regeneration rate during the osteogenesis process was evaluated using sequential fluorescent labeling and the results are shown in Fig. 8d and e. Compared with the TCP group, both the TCP-PDA/AL and TCP-PDA/AL-IL-4 groups exhibited a significantly wider double fluorescence spacing and a markedly increased bone deposition rate. Notably, the TCP-PDA/AL-IL-4 group showed the fastest mineral deposition rate at 3.06 μm/day, indicating enhanced bone conversion efficiency and accelerated bone regeneration, corroborating with the micro-CT findings.

The osteoclast activity following the 6-week material implantation was evaluated using TRAP staining and the results are shown in Fig. 8f. Osteoclasts were observed in all three groups, with the TCP-PDA/AL-IL-4 group exhibiting the lowest number of osteoclasts. This result can be attributed to the continuous release of AL, which inhibits osteoclast activity, and IL-4, which suppresses osteoclast formation by inhibiting TNF signaling and blocking RANKL-dependent activation of the NF-κB signaling pathway [47]. Consistent with the results of in vitro cell experiments, the results of implantation in vivo indicated that the TCP-PDA/AL-IL-4 group, with its sequential modulation function, effectively inhibits osteoclast formation, thereby reducing bone resorption and promoting bone regeneration.

4. Conclusion

Functionalized bioceramic scaffolds with sequentially regulating immune-osteogenesis were successfully constructed. The initial release of IL-4 facilitates the polarization of macrophages toward the M2 phenotype, thereby mitigating inflammatory responses and establishing a conducive osteogenic immune microenvironment. Subsequently, the sustained release of AL promotes osteogenic differentiation, inhibits osteoclast activity, and reduces bone resorption. The sequential modulation significantly increased new bone formation within the bioceramic scaffold and enhanced the mineral deposition rate, accelerating bone regeneration in an osteoporotic environment.

CRediT authorship contribution statement

Yan Yang: Writing – original draft, Methodology, Investigation, Formal analysis, Data curation, Conceptualization. Jiaqian Zheng: Methodology, Formal analysis, Conceptualization. Qiji Lu: Supervision, Methodology, Formal analysis. Yudi Kuang: Supervision, Methodology, Formal analysis. Jingjing Diao: Writing – review & editing, Supervision, Resources, Project administration, Methodology, Funding acquisition, Conceptualization. Naru Zhao: Writing – review & editing, Supervision, Resources, Project administration, Funding acquisition, Conceptualization.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Appendix A. Supplementary data

The following is the supplementary data to this article:

Data availability

Data will be made available on request.