Adenovirus-Mediated Expression of BMP-2 and BFGF in Bone Marrow Mesenchymal Stem Cells Combined with Demineralized Bone Matrix For Repair of Femoral Head Osteonecrosis in Beagle Dogs

Abstract

Background: This study investigated the effect of using adenovirus-mediated expression of bone morphogenetic protein 2 (Ad-BMP-2) and basic fibroblast growth factor (bFGF) in bone marrow mesenchymal stem cells (BMSCs) in combination with a demineralized bone matrix (DBM) to repair osteonecrosis of the femoral head (ONFH) in Beagle dogs. Methods: A total of 30 Beagle dogs were selected for the isolation of BMSCs, which were cultured and transfected with the recombinant adenovirus vector Ad-BMP2-bFGF-GFP (carrying BMP-2 and bFGF) or a control adenovirus plasmid (encoding green fluorescent protein (Ad-GFP)). The expression of the transfected BMP-2 and bFGF proteins was detected by Western blotting. After transfection, the BMSCs were induced to undergo osteoblastic differentiation. The DBM was prepared to construct a DBM/BMSC complex. Beagle models of canine femoral head defects and necrosis were established and divided into control, DBM, DBM/BMSC, vector Ad-BMP2-bFGF-GFP and Ad-GFP groups. The composite graft was then implanted, and new bone morphology was visualized via X-ray at 3, 6 and 12 weeks after the operation. Hematoxylin and eosin (HE) staining and Masson’s trichrome staining were used to identify new bone formation. Immunohistochemistry was performed to calculate the density of new blood vessels. The compressive and bending strength of the BMSCs was evaluated at 12 weeks after the operation. Results: BMSCs were successfully isolated. The protein expression of BMP-2 and bFGF was significantly higher in the Ad-BMP-2/bFGF group than the normal and Ad-GFP groups. Compared with the control group, at 12 weeks after the operation, the DBM, DBM/BMSC, vector Ad-BMP2-bFGF-GFP and Ad-GFP groups showed a larger area of new bone, higher X-ray scores, greater neovascularization density, and increased compressive and bending strength. The most significant modifications occurred in thevector Ad-BMP2-bFGF-GFP group. Conclusion: The results indicate that the use of Ad-BMP-2/bFGF-modified BMSCs in conjunction with DBM could successfully repair ONFH in a dog model by promoting bone formation and angiogenesis.

Sequential application of bFGF and BMP‐2 facilitates osteogenic differentiation of human periodontal ligament stem cells

“…bFGF could stimulate cell motility via activating the FGFR1‐PI3K‐Rac1 pathway and promoted osteogenic differentiation through the activation of MAPK and smad‐dependent signaling pathway . The current application modalities of bFGF and BMP‐2 were designed to release the two factors at the same time . Our study confirmed that sequential application of bFGF and BMP‐2 showed more excellent effect in up‐regulating the osteogenic capability of PDLSCs when compared with the simultaneous application manner.…”

“…Basing on current studies, combination of bFGF and BMP‐2 may have broad applications in bone regeneration medicine. It has been reported that the combination of bFGF and BMP‐2 could induce stem cells osteogenic differentiation . The current application of these two growth factors can be summarized to three types.…”

“…Secondly, transfect stem cells with adenoviral vector carrying bFGF or BMP‐2 to defective area to continuously produce the proteins . Thirdly, implant biological materials which loaded the transfected stem cells with the overexpression of bFGF and BMP‐2 genes into bone defect area . However, the aforementioned application modalities of bFGF and BMP‐2 released the two factors at the same time, which is not appropriate for the real state of tissue regeneration.…”

“…It has been reported that the combination of bFGF and BMP-2 could induce stem cells osteogenic differentiation. [28][29][30] The current application of these two growth factors can be summarized to three types. Firstly, apply directly to promote endogenous stem cells proliferation and differentiation.…”

Stromal-Cell-Derived Factor (SDF) 1-Alpha Overexpression Promotes Bone Regeneration by Osteogenesis and Angiogenesis in Osteonecrosis of the Femoral Head

“…The latter risk was alleviated by rifampicin likely through enhancing P-gp activity [20], whereas glucocorticoid-induced ONFH was successfully prevented by treatment with Vitamin K2 [21]. Moreover, augment the expression of BMP-2 and BFGF in BMSCs promoted bone repair of ONFH [22]. These results suggest that multiple factors are involved in the therapeutic effects of BMSCs on ONFH, and additional treatment with drug or transgenic modification may promote their efficacy.…”