Runx2-Modified Adipose-Derived Stem Cells Promote Tendon Graft Integration in Anterior Cruciate Ligament Reconstruction.

Xin Zhang,Jiying Zhang,Xiaoqing Hu,Linghui Dai,Chunyan Zhou,Xin Fu,Qiang Liu,Yong Ma,Wenqing Chen,Ping Chen,Yingfang Ao,Yanbin Pi,Zhenxing Shao,Xiaoning Duan

doi:10.1038/srep19073

Abstract

Runx2 is a powerful osteo-inductive factor and adipose-derived stem cells (ADSCs) are multipotent. However, it is unknown whether Runx2-overexpressing ADSCs (Runx2-ADSCs) could promote anterior cruciate ligament (ACL) reconstruction. We evaluated the effect of Runx2-ADSCs on ACL reconstruction in vitro and in vivo. mRNA expressions of osteocalcin (OCN), bone sialoprotein (BSP) and collagen I (COLI) increased over time in Runx2-ADSCs. Runx2 overexpression inhibited LPL and PPARγ mRNA expressions. Runx2 induced alkaline phosphatase activity markedly. In nude mice injected with Runx2-ADSCs, promoted bone formation was detected by X-rays 8 weeks after injection. The healing of tendon-to-bone in a rabbit model of ACL reconstruction treated with Runx2-ADSCs, fibrin glue only and an RNAi targeting Runx2, was evaluated with CT 3D reconstruction, histological analysis and biomechanical methods. CT showed a greater degree of new bone formation around the bone tunnel in the group treated with Runx2-ADSCs compared with the fibrin glue group and RNAi Runx2 group. Histology showed that treatment with Runx2-ADSCs led to a rapid and significant increase at the tendon-to-bone compared with the control groups. Biomechanical tests demonstrated higher tendon pullout strength in the Runx2-ADSCs group at early time points. The healing of the attachment in ACL reconstruction was enhanced by Runx2-ADSCs.

Highlights

Adipose-derived stem cells (ADSCs), a type of adult mesenchymal stem cell, are capable of self-renewal and increasing proliferation
adipose-derived stem cells (ADSCs) pellets were cultured in standard chondrogenic differentiation medium for 14 days, after which they were stained with toluidine blue to indicate the secretion of proteoglycan (Fig. 1c)
Bone morphogenetic protein (BMP) is a member of transforming growth factor-β (TGF-β ) family, with more than 10 members involved in osteoblast differentiation and bone formation regulation[8]

Summary

Introduction

Adipose-derived stem cells (ADSCs), a type of adult mesenchymal stem cell, are capable of self-renewal and increasing proliferation. The Runx[2] gene was first cloned in 1997 and is exclusively expressed in bone tissue or osteoblasts[5]. Runx[2] is an osteoblast-specific transcription activator. Ossification and osteoblasts formation are impaired in Runx[2] gene-deficient neonatal mice (Runx2−/−), as shown by X-ray and histological analysis. The cis-element in the OCN gene promoter is OES2, which has an identical sequence to the Runt-binding site of Runx[27]. Runx[2] regulates the expression of osteoblast-specific genes. We hypothesized that Runx[2] enhances the osteogenic differentiation of ADSCs and the tendon-to-bone healing in ACL reconstruction. We found that ADSCs overexpressing Runx[2] induced osteogenic differentiation in vitro and extensively regenerated new bone tissue at the transplantation site. In a rabbit ACL reconstruction model, with a slow-release fibrin glue matrix to deliver Runx2-ADSCs, we demonstrated that this material accelerated tendon-to-bone integration early after ACL reconstruction

Objectives

Methods

Results

Conclusion