The construction of a novel xenograft bovine bone scaffold, (DSS)6-liposome/CKIP-1 siRNA/calcine bone and its osteogenesis evaluation on skull defect in rats

Gang Xu; Xiantong Hu; Liwei Han; Yantao Zhao; Zhonghai Li

doi:10.1016/j.jot.2021.02.001

The construction of a novel xenograft bovine bone scaffold, (DSS)6-liposome/CKIP-1 siRNA/calcine bone and its osteogenesis evaluation on skull defect in rats

J Orthop Translat. 2021 Mar 2:28:74-82. doi: 10.1016/j.jot.2021.02.001. eCollection 2021 May.

Authors

Gang Xu^{1

2}, Xiantong Hu^{3

4}, Liwei Han^{3

4}, Yantao Zhao^{3

4}, Zhonghai Li^{1

2}

Affiliations

¹ Department of Orthopaedics, First Affiliated Hospital of Dalian Medical University, Dalian, 116011, PR China.
² Key Laboratory of Molecular Mechanism for Repair and Remodeling of Orthopaedic Diseases, Liaoning Province, Dalian, 116011, PR China.
³ Department of Orthopaedics, Fourth Medical Center of PLA General Hospital, Beijing, 100048, PR China.
⁴ Beijing Engineering Research Center of Orthopaedic Implants, Beijing, 100048, PR China.

Abstract

Background: Xenograft bone scaffolds have advantages such as mechanical strength, sufficient source and safety. Combined with siRNA properly targeting CKIP-1, a negative regulator of osteogenesis, may contribute to the repair result of calcine bone alone.

Methods: Herein, we constructed a novel xenograft bovine bone scaffold namely (DSS)6-liposome/CKIP-1 siRNA/calcine bone, the characteristics of which were investigated by confirming the effect of (DSS)6-liposome, observing the appearance and testing mechanical strength of calcine bone, and observing the combined result of CKIP-1 siRNA by FAM immunofluorescence. In addition, cytotoxicity by CCK-8 and LDH activity of L929 cells and MC3T3-E1 osteoblasts cultured with the scaffold were tested in vitro, primary osteoblasts proliferation, the mRNA expressions of CKIP-1, ALP, COL1-α and OCN, the protein expressions of CKIP-1, BMP-2, COL-1 and Runx2 and calcium nodules were also determined by CCK-8, RT-qPCR, western-blot and Alizarin Red staining in vitro. Then, we successively established the skull defect model for evaluating the repair result of the novel scaffold by HE staining of 2, 4, 8 and 12 weeks, immumohistochemical stainings of 2, 4, 8 and 12 weeks such as ALP, COL-1α and OCN, Mirco-CT scanning of 4 and 12 weeks and the relative parameters and so on in vivo.

Results: It indicated that (DSS)6-liposome/CKIP-1 siRNA/calcine bone could successfully knock down the CKIP-1 mRNA and protein expressions, promote osteoblasts proliferation with the little cytotoxicity in vitro, increase the protein expressions of BMP-2, COL-1 and Runx2 in vitro, increase mRNA expressions of ALP, COL-1α and OCN in vitro and in vivo, and have a better bone defect repair effect with few side effects in rats after 12 weeks.

Conclusion: Our research indicates (DSS)6-liposome/CKIP-1 siRNA/calcine bone could repair skull defects well in rats, and it may lay the foundation of applicating the novel xenograft bone scaffold in the clinical.

The translational potential of this article: These findings provide evidence that (DSS)6- liposome/CKIP-1 siRNA/calcine bone could be used as a novel xenograft bone scaffold for osteogenesis with the good safety.

Keywords: Bone defect; True bone calcine; Xenograft bone scaffolds; casein kinase 2 interacting protein-1; siRNA.