Aspirin synergizes with mineral particle-coated macroporous scaffolds for bone regeneration through immunomodulation

Introduction:: Calcium phosphate (CaP) particles have been widely used to enhance bone repair due to their osteoconductive cues. However, CaP can also cause undesirable inflammatory responses and lead to bone resorption.¹ As such, it is important to understand the effect of varying composition and size of CaP particles on macrophage (Mφ) polarization to minimize inflammation while leveraging their osteoconductive benefits. Aspirin is a nonsteroid anti-inflammatory drug that is widely used in clinical settings, and previous studies have shown the benefit of aspirin (Asp) on immunomodulation and bone formation.² The goals of this study are to determine the optimal CaP particle composition and size that is least inflammatory while promoting MSC osteogenesis and assess the synergy between Asp with optimized CaP particle on microporous scaffold-mediated bone regeneration in a critical size cranial defect model. We have chosen a gelatin-based microribbon (µRB) scaffold due to its macroporosity and advantages over nanoporous hydrogels in supporting bone formation in vivo.³ We hypothesize that varying CaP particle composition and size would modulate macrophage inflammatory responses in vitro, and optimized CaP particles would synergize with Asp to accelerate endogenous bone regeneration in a critical-size cranial defect model.

Materials and Methods:: A total of four CaP particles were evaluated with varying composition hydroxyapatite (HA) v.s. tricalcium phosphate (TCP) or size (nano- or micro). All CaP was incorporated into gelatin µRB scaffolds (3.5% w/v) through physical adsorption. A Mφ cell line (RAW) was seeded on top of CaP-µRB scaffolds for 18 h, and phenotypes were evaluated by qPCR of M1 and M2 markers. Conditioned medium (CM) of Mφ cultured on different scaffolds was also used to treat MSCs in µRB scaffolds with the same CaP cues. Bone formation was evaluated using alizarin red S (ARS) staining at day 16 (Fig. 1A). The lead CaP particle was further combined with varying concentrations of Asp to assess their effects on MSC osteogenesis, osteoclast activity, and Mφ polarization in vitro. Outcomes were evaluated by staining of osteocalcin (OCN) and TRAP, as well as ELISA. Finally, the efficacy of µRB scaffolds with optimized CaP particle and Asp for enhancing bone formation was tested in a mouse critical-sized cranial defect model. Bone formation was monitored by µCT for up to 6 weeks.

Results, Conclusions, and Discussions:: Results: We identified mTCP as the lead CaP particle, which did not increase proinflammatory cytokine production (TNF-α/M1), while promoting anti-inflammatory cytokines (IL10/M2) (Fig. 1B). µRB scaffolds with mTCP also resulted in the most robust mineralization, and Mφ CM from mTCP showed the least inhibitory effects on MSC bone formation (Fig. 1C). Adding Asp inhibited M1 Mφ polarization, decreased OC activity and enhanced MSC osteogenesis in mTCP scaffolds in a dosage-dependent manner (Fig. 1D-F). When transplanted in vivo, the mTCP+20 µg Asp group led to rapid endogenous bone regeneration, with bone defects filled completely as early as week 2, with no bone resorption by week 6. In contrast, mTCP with no Asp or 10 µg Asp only led to 12% or 19% bone volume filled by week 2, followed by further bone resorption by week 6. (Fig. 1G-H).

Conclusion: We demonstrate Asp and TCP microparticles synergize to enhance macroporous µRB scaffold-mediated bone regeneration by promoting MSC osteogenesis and reducing inflammation and osteoclast activity. The findings of this study may guide the rational design of scaffolds with CaP particles and Asp as a cell-free therapy for treating critical-sized bone defects.


Acknowledgements (Optional): : The authors acknowledge NIH R01DE024772, R01AR074502, Stanford MCHRI Postdoctoral Fellowship, NSF GRFP, Stanford BioX fellowship, and Stanford Bioeng REU for funding.


References (Optional): : [1] V.Frédéric, et al. Acta biomaterialia 9.2 (2013): 4956-4963.
[2] Y. Xie, et al. Cell Biosci 2019, 9, 103.
[3] L. H. Han, J Biomed Mater Res A 2016, 104, 1321.