Microribbon Hydrogels with Tunable Compositions Enhances Bone Regeneration through Immunomodulation

Introduction:: Previous research on tissue engineering for bone regeneration largely focuses on promoting stem cell osteogenesis.¹ Recent studies have highlighted the important roles of immune cells in bone regeneration.² Especially, the transition from M1 to M2 macrophage phenotype is considered essential for proper bone healing. Recent studies showed tuning hydrogel compositions can enhance tissue regeneration through immunomodulation, but previous work was limited to nanoporous hydrogels and soft tissues.³ Our lab has recently reported gelatin-based microribbon (µRB) scaffolds as 3D macroporous cell niches that support mesenchymal stem cell (MSC)-based bone formation in vivo.⁴ The goal of this study is to develop µRB scaffolds with tunable compositions to modulate MSC/macrophage (Mφ) crosstalk and enhance bone regeneration through immunomodulation. We hypothesize that adding chondroitin sulfate (CS) into gelatin µRB would modulate MSC osteogenesis and Mφ polarization in a dose-dependent manner, and optimal composition that supports bone formation in a 3D mixed MSC/ Mφ co-culture model in vitro would accelerate cranial bone formation in vivo.

Materials and Methods:: A total of seven µRB formulations were synthesized with varying ratios of Gel/CS (100:0, 99:1, 95:5, 90:10, 50:50, and 25:75) by wet spinning. Scaffold morphology was characterized by scanning electron microscope (SEM). The effects of tuning µRB composition on MSC/ Mφ phenotype and bone formation were assessed in vitro using 3D mono-culture and mixed co-culture models. Alizarin red S (ARS) and trichrome staining were performed to assess mineral and collagen deposition in vitro. Mφ polarization was determined by qPCR of M1 and M2 markers. The selected leading Gel/CS composition was further tested in a mouse critical-sized cranial defect model. The effects of tuning µRB composition on immunomodulation in vivo were evaluated by flow cytometry and single-cell RNA sequencing (scRNAseq) at week 1. Bone formation was monitored by µCT up to week 6.


Results, Conclusions, and Discussions:: Results: Increasing the CS ratio enhanced MSC osteogenesis and mineralized bone formation in µRB in mono-culture (Fig.1A). However, increasing the CS ratio beyond 50% significantly enhanced Mφ polarization towards proinflammatory M1 (Fig.1B). Using a 3D mixed co-culture model, we identified Gel50/CS50 as the lead formulation that led to the most robust bone formation in vitro (Fig.1C). This trend was further validated in vivo. Gel50/CS50 group led to rapid endogenous bone regeneration, with almost 95% bone defects filled by week 6 (Fig.1D). Flow cytometry analyses showed Gel50/CS50 promoted Mφ polarization towards pro-regenerative phenotype, and accelerated early MSC recruitment (Fig.1E). ScRNAseq further revealed Gel50/CS50 resulted in increased recruitment of an M2 Mφ subpopulation with unique markers, which is associated with the enhanced bone formation (Fig.1F).

Conclusion: We demonstrate tuning Gel/CS ratio in macroporous µRB hydrogels as a promising strategy to enhance endogenous bone regeneration in a critical-sized defect through immunomodulation. The 3D MSC/Macrophage co-culture model was effective in identifying optimal scaffold composition that induces regenerative immunomodulation in vivo. This work fills a critical gap of knowledge on tuning compositions of macroporous hydrogels on immunomodulation in critical-sized cranial defects and may be broadly applied to enhance the regeneration of other tissue types.


Acknowledgements (Optional): : The authors acknowledge NIH R01DE024772 (F. Y.), R01AR074502 (FY), Stanford Maternal and Children’s Health Institute Postdoctoral Fellowship (N.S.), NSF GRFP (CV), EDGE fellowship (CV) and Stanford Bioeng REU (PS) for funding.


References (Optional): : [1] S. Yin, W. Zhang, Z. Zhang, X. Jiang, Adv Healthc Mater 2019, 8, e1801433.
[2] N. Su, C. Villicana, F. Yang, Biomaterials 2022, 121604.
[3] K. Sadtler, M. T. Wolf, S. Ganguly, C. A. Moad, L. Chung, S. Majumdar, F. Housseau, D. M. Pardoll, J. H. Elisseeff, Biomaterials 2019, 192, 405. [4] L. H. Han, B. Conrad, M. T. Chung, L. Deveza, X. Jiang, A. Wang, M. J. Butte, M. T. Longaker, D. Wan, F. Yang, J Biomed Mater Res A 2016, 104, 1321.