Modulating immune-stem cell crosstalk enables robust bone regeneration via tuning compositions of macroporous scaffolds

Abstract

Following bone injury, macrophages (Mφ) initiate the immune response by secreting signals that recruit mesenchymal stem cells (MSC) and other niche cells to shape healing. Despite its importance, the potential of enhancing bone regeneration by modulating immune-stem cell crosstalk is largely unexplored. Here, we report a macroporous microribbon (µRB) scaffold with tunable ratios of gelatin (Gel) and chondroitin sulfate (CS), achieving rapid endogenous bone regeneration in a critical-sized defect model without exogenous growth factors or cells. The 3D MSC/Mφ co-culture model, but not the mono-culture model, effectively identified Gel50_CS50 as the leading ratio for accelerating bone regeneration in vivo. Single-cell sequencing (scRNAseq) and CellChat analysis revealed that Gel50_CS50 significantly enhanced the cellular crosstalk among Mφ and other bone niche cell types, with signaling pathways linked to anti-inflammation, angiogenesis, and osteogenesis. This study demonstrates Gel50_CS50 µRB as a promising biomaterial-based therapy for treating critical-sized bone defects by modulating cellular crosstalk.

Fig. 1. MSC- Mφ co-culture model identified Gel50_CS50 as the optimal ratio for bone formation in vitro.

A SEM and 3D reconstruction of µRB scaffolds; Scale bar: 100 µm. B–D Assessment of osteogenic gene expression (B, C) and calcium deposition (D) in MSC mono-culture and MSC- Mφ coculture; Scale bar in (D): 200 µm. Data are represented as mean ± S.D. (n = 3/group). **P < 0.01, ***P < 0.001, ****P < 0.0001.

Fig. 2. Gel50_CS50 significantly accelerated endogenous bone regeneration in vivo, consistent with the prediction of the Mφ-MSC co-culture in vitro model.

A Representative μCT images. B Quantitative characterization of bone volume/total volume (BV/TV) and Bone mineral density (BMD), n = 5/group. C, D HE and Trichrome histology of cranial bone defect area at week 6; Scale bar in (C, D): 500 µm. E Representative image and quantification of angiogenesis using CD31 immunostaining at week 6; Scale bar: 100 µm; n = 3/group. F, G Percentage of recruited MSC (CD90+) (F) using flow cytometry and representative dot plots (G) at day 7 in the cranial bone defects; n = 3/group. Data are represented as mean ± S.D. *P < 0.0001, ****P < 0.0001.

Fig. 3. High CS ratios significantly promoted M1 Mφ polarization, whereas Gel50_CS50 co-spun µRBs favored more M2 Mφ polarization in vitro and in vivo.

A–D Assessment of TNF-α and IL-10 cytokine secretion in Mφ mono-culture (A, B) and MSC- Mφ coculture (C, D) (n = 3/group). E, F Percentage of M1 Mφ (CD11b + F4/80 + CD86+) population (E), M2 Mφ (CD11b+F4/80 + CD206+) population (F) in the defect at day 7, characterized by flow cytometry. G Representative flow cytometry histogram of (E, F). Data are represented as mean ± S.D. (n = 3/group). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Fig. 4. Gel50_CS50 enriched a unique subpopulation of M2-like Mφ in vivo.

A Recruited cell composition in macroporous scaffolds determined by single-cell RNA sequencing (RNA-Seq) at day 7 after bone injury. B, C Key gene markers (B) and enriched GO (C) among three types of Mφ. D Percentage of each Mφ subpopulation in different material composition. E Validation of three types of Mφ by flow cytometry staining. F Validation of M2-like 2 gene markers induced by Gel50_CS50 using Mφ mono-culture model in vitro; Data are represented as mean ± S.D. (n = 3/group). **P < 0.01, ****P < 0.0001.

Fig. 5. Gel50_CS50 µRB scaffolds exhibited robust cellular crosstalk and differential crosstalk patterns in vivo.

A, B Circle plots revealed robust cellular crosstalk among varying cell populations, which was further enriched in Gel50_CS50 composition. C Outgoing and incoming interactions were mediated by different cell populations in response to varying material compositions. D, E Differential signaling pathways (D) and crosstalk patterns (E) featured by Gel100 and Gel50_CS50.

Fig. 6. Gel50_CS50 µRB scaffolds facilitated to Mφ crosstalk in vivo.

A Differential signaling pathways from Mφ to all other cell populations featured by Gel100 and Gel50_CS50 scaffolds. B–F Representative circle plots highlighting crosstalk patterns of differential signaling pathways.

Fig. 7. Mechanism of Gel50_CS50 composition induced robust bone regeneration through immune-bone niche crosstalk.

The schematic was created using bioRender.