Bone-Targeting Liposome-Encapsulated Salvianic Acid A Improves Nonunion Healing Through the Regulation of HDAC3-Mediated Endochondral Ossification

Abstract

Aim

Nonunion is a major complication in fracture repair and remains a challenge in orthopaedics and trauma surgery. In this study, we aimed to evaluate the effectiveness of treatment of nonunion with a large radial defect using a bone-targeting liposome-encapsulated salvianic acid A (SAA-BTL)-incorporated collagen sponge and further elucidate whether the effects were closely related to histone deacetylase 3 (HDAC 3)-mediated endochondral ossification in nonunion healing process.

Methods

Fifteen New Zealand female rabbits were randomly divided into three groups. Segmental radius critical size defects (15 mm) were created via surgery on both the forelimbs of the rabbits. The SAA-BTL/SAA/saline-incorporated collagen sponges were implanted into the defects in the three groups, respectively, for four weeks of treatment. X-ray imaging, micro-computed tomography (CT) analysis, histology, and immunofluorescence analysis (HDAC3, collagen II, VEGFA, and osteocalcin) were performed to determine the effects of the treatments. In addition, a short interfering RNA was applied to induce HDAC3 knockdown in the chondrogenic cell line ATDC5 to investigate the roles of HDAC3 and SAA intervention in endochondral ossification in nonunion healing.

Results

X-ray imaging and micro-CT results revealed that SAA-BTL-incorporated collagen sponges significantly stimulated bone formation in the nonunion defect rabbit model. Furthermore, immunofluorescence double staining and histology analysis confirmed that SAA-BTL significantly increased the expression of P-HDAC3, collagen II, RUNX2, VEGFA, and osteocalcin in vivo; accelerated endochondral ossification turnover from cartilage to bone; and promoted long bone healing of nonunion defects. ATDC5 cells knocked down for HDAC3 showed significantly decreased expression of HDAC3, which resulted in reduced expression of chondrogenesis, osteogenesis, and angiogenesis biomarker genes (Sox9, Col10a1, VEGFA, RUNX2, and Col1a1), and increased expression of extracellular matrix degradation marker (MMP13). SAA treatment reversed these effects in the HDAC3 knockdown cell model.

Conclusion

SAA-BTL can improve nonunion healing through the regulation of HDAC3-mediated endochondral ossification.

Keywords:

• nonunion
• fracture
• HDAC3
• endochondral ossification
• bone-targeting liposome
• salvianic acid A

Acknowledgments

This study was supported in part by the National Natural Science Foundation of China (No. 81703584), Guangdong Province Natural Science Foundation of China (Nos. 2017A030310614, 2016B030309002, 2018A030307036), Science and Technology Competitive Financial Fund of Zhanjiang (Nos. 2016A01012, 2017A01021, 2017A06012), “Group-type” Special Support Project for Education Talents in Universities (4SG19045G), Shenzhen Dapeng New Area Industrial Development Special Fund Project (KY20180104), and Guangdong Medical University Scientific Research Fund (No. B2017001).

Author Contributions

All authors meet the criteria for authorship. All authors made substantial contributions to conception and design of the study, acquisition of data, and analysis and interpretation of data; took part in drafting the article or revising it critically for important intellectual content; gave final approval of the version to be published; and agree to be accountable for all aspects of the work.

Disclosure

The authors declare no conflicts of interest.