Adenovirus-mediated transfer of siRNA against Runx2/Cbfa1 inhibits the formation of heterotopic ossification in animal model

doi:10.1016/j.bbrc.2006.08.089

Biochemical and Biophysical Research Communications

Volume 349, Issue 2, 20 October 2006, Pages 564-572

https://doi.org/10.1016/j.bbrc.2006.08.089 Get rights and content

Abstract

The heterotopic ossification of muscles, tendons, and ligaments is a common problem faced by orthopaedic surgeons. Runx2/Cbfa1 plays an essential role during the osteoblast differentiation and is considered as a molecular switch in osteoblast biology. RNA interference technology is a powerful tool for silencing endogenous or exogenous genes in mammalian cells. In this study, we investigated the effect of Runx2/Cbfa1-specific siRNA on osteoblast differentiation and mineralization in osteoblastic cells, and then constructed adenovirus containing siRNA against Runx2/Cbfa1 (Ad-Runx2-siRNA) to inhibit the formation of heterotopic ossification induced by BMP4, demineralized bone matrix, and trauma in animal model. Our results showed that the Runx2/Cbfa1-specific siRNA could inhibit the expression of Runx2/Cbfa1 at the level of mRNA and protein. Analysis of the expression of osteoblast maturation genes including type I collagen, osteopontin, bone sialoprotein, and osteocalcin, alkaline phosphatase activity, and matrix mineralization (von kossa) revealed that osteoblast differentiation was inhibited in cultured primary mouse osteoblasts transduced with Ad-Runx2-siRNA. Furthermore, adenovirus-mediated transfer of siRNA against Runx2/Cbfa1 could inhibit the formation of heterotopic ossification induced by BMP4, demineralized bone matrix, and trauma in animal model. It is likely that the inhibition of Runx2/Cbfa1 by RNAi could be developed as a powerful approach to prevent or treat heterotopic ossification.

Section snippets

Cell culture

Primary osteoblasts were isolated from the calvariae of 21-day fetal mice as previously described [35]. In brief, cells were obtained from the calvariae (the dura and periosteum removed) by five sequential digestions of 10 min at 37 °C in phosphate-buffered saline (PBS) containing 0.05% collagenase. Cells from the fourth and fifth digests were used in the present study. MC3T3-E1 was obtained from Chinese Academy of Medical Sciences (Peking, China). Cells were suspended in Dulbecco’s modified

Transfection of siRNA against Runx2 strongly suppressed Runx2 expression in MC3T3-E1 cells

We synthesized five siRNA against different target sites of Runx2, and the concentration of the siRNA preparation was assessed by measuring the absorbance of the siRNA sample at 260 nM with UV spectrophotometer. The degree of purification of siRNA was assessed by gel electrophoresis on 2% agarose that showed a single band at 21 bp according to the Ambion’s manufacturer (data not shown). We then transfected siRNA against Runx2, as well as scrambled siRNA, separately into MC3T3 cells by

Discussion

The pathogenesis of heterotopic ossification is unclear, but may involve inappropriate differentiation of pluripotent mesenchymal cells into bone forming cells under the influence of skeletal growth factors [18]. Osteoblasts differentiate from pluripotent precursor cells that have the capacity to become adipocytes, skeletal muscle cells, tendon, or fibroblasts [37], [38]. During differentiation, a program of gene expression occurs that is characterized by sequential steps of proliferation,

Acknowledgment

This research was supported by the program of gene therapy on sports injury sponsored by the State Sports General Administration of PR China.

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Osterix (OSX), as a downstream gene of RUNX2, is also implicated in the tooth development [34,35]. Osteocalcin (OCN), a specific biochemical marker of osteoblast function, can reflect the activity of osteoblast [36]. Dentin sialophosphoprotein (DSPP) can encode protein DSP and DPP as major non-collagen protein in dentin matrix.
Periodontal ligament stem cells (PDLSCs) are vital for the regeneration of periodontal tissues. Potassium dihydrogen phosphate (KH₂PO₄) has recently been applied as a component of the mineralization inducing medium (MM), which can be used to induce osteogenic differentiation of dental stem cells. However, whether KH₂PO₄ has effects on PDLSCs has not been studied.
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ALP activity assay and ALP protein expression examination revealed that 1.8 mmol/L KH₂PO₄ was the optimal concentration for the induction of hPDLSCs by KH₂PO₄. The proliferation and mineralization capacity of PDLSCs treated with KH₂PO₄ were enhanced as compared with the control group. PDLSCs treated with KH₂PO₄ showed an improved proliferation capacity in logarithmic growth phase at day 7. As PDLSCs were treated with KH₂PO₄, the expression of odonto/osteogenic markers (OCN/OCN, DSP/DSPP, OSX/OSX, RUNX2/RUNX2, and ALP/ALP) in cells were up-regulated at day 3 or 7. Moreover, the expression of IκBα in cytoplasm was down-regulated, along with an increased expression of p-P65 in cytoplasm and an up-regulated expression of P65 in nucleus. When treated with BMS345541 (the specific NF-κB inhibitor), the odonto/osteogenic differentiation of KH₂PO₄-treated PDLSCs was significantly attenuated.
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Adenovirus-mediated transfer of siRNA against Runx2/Cbfa1 inhibits the formation of heterotopic ossification in animal model

Abstract

Section snippets

Cell culture

Transfection of siRNA against Runx2 strongly suppressed Runx2 expression in MC3T3-E1 cells

Discussion

Acknowledgment

Burns

Cell

Cell

Cell

Cell

Biochem. Biophys. Res. Commun.

Osteoarthr. Cartilage.

Heterotopic ossification about the hip after intramedullary nailing for fractures of the femur

J. Bone Joint Surg. [Am.]

Ectopic bone formation following low friction arthroplasty of the hip

Clin. Orthop.

Ectopic ossification following total hip arthroplasty: is diffuse idiopathic skeletal hyperostosis a risk factor?

Br. J. Rheumatol.

Bone formation following carpal interposition arthroplasty

Skeletal Radiol.

Ectopic bone formation after total hip arthroplasty

Clin. Orthop.

Pathologic bone formation

Clin. Orthop.

Turnbuckle orthotic correction of elbow-flexion contractures after acute injuries

J. Bone. Joint. Surg. [Am.]

Treatment of ectopic ossification about the elbow

Clin. Orthop.

Heterotopic ossification

Orthopedics

Fibrodysplasia ossificans progressiva. The clinical features and natural history of 34 patients

J. Bone. Joint. Surg. [Br.]

Periarticular heterotopic ossification after total hip arthroplasty, Risk factors and consequences

Clin. Orthop.

Heterotopic ossification: are range of motion exercises contraindicated?

J. Burn. Care. Rehabil.

Postburn heterotopic ossification: insights for management decision making

J. Trauma.

Impact of clinically significant heterotopic ossification on functional outcome after traumatic brain injury

J. Head. Trauma. Rehabil.

Risk factors for heterotopic ossification in spinal cord injury

Arch. Phys. Med. Rehabil.

Heterotopic bone formation in the gastrointestinal tract

Arch. Pathol. Lab. Med.

Observations on the induction of bone in soft tissues

J. Bone. Joint. Surg. [Br.]

Bone: formation by autoinduction

Science

Ex vivo gene therapy to produce bone using different cell types

Clin. Orthop

Differential expression of bone and cartilage related genes in fibrodysplasia ossificans progressiva, myositis ossificans traumatica, and osteogenic sarcoma

Clin. Orthop.