Abstract
Selection of appropriate osteoinductive growth factors, suitable delivery method and proper supportive scaffold are critical for a successful outcome in bone tissue engineering using bone marrow stromal cells (BMSC). This study examined the molecular and functional effect of a combination of adenoviral mediated expression of bone morphogenetic protein-2 (BMP2) in BMSC and recently developed and characterized, biodegradable Poly(L-lactide-co-є-caprolactone){poly(LLA-co-CL)}scaffolds in osteogenic molecular changes and ectopic bone formation by using in vitro and in vivo approaches. Pathway-focused custom PCR array, validation using TaqMan based quantitative RT-PCR (qRT-PCR) and ALP staining showed significant up-regulation of several osteogenic and angiogenic molecules, including ALPL and RUNX2 in ad-BMP2 BMSC group grown in poly(LLA-co-CL) scaffolds both at 3 and 14 days. Micro CT and histological analyses of the subcutaneously implanted scaffolds in NOD/SCID mice revealed significantly increased radiopaque areas, percentage bone volume and formation of vital bone in ad-BMP2 scaffolds as compared to the control groups both at 2 and 8 weeks. The increased bone formation in the ad-BMP2 group in vivo was paralleled at the molecular level with concomitant over-expression of a number of osteogenic and angiogenic genes including ALPL, RUNX2, SPP1, ANGPT1. The increased bone formation in ad-BMP2 explants was not found to be associated with enhanced endochondral activity as evidenced by qRT-PCR (SOX9 and FGF2) and Safranin O staining. Taken together, combination of adenoviral mediated BMP-2 expression in BMSC grown in the newly developed poly(LLA-co-CL) scaffolds induced expression of osteogenic markers and enhanced bone formation in vivo.
Highlights
Bone regeneration using tissue engineering approach, which utilizes mesenchymal stem cells grown in an osteoconductive scaffold and with osteoinductive growth factors is a promising approach and has been studied for over a decade [1]
bone marrow stromal cells (BMSC) transduced with Adenoviral vector carrying only eGFPcoding sequences (ad-GFP) and ad-Bone Morphogenetic protein-2 (BMP2) viral particles demonstrated similar morphology in monoculture (Fig 1A–1D). ad-BMP2 BMSC grown in monolayer expressed significantly doi:10.1371/journal.pone.0147507.g001
Alkaline phosphatase (ALP) level was found to be significantly induced at day 3 and the elevated level was maintained at day 14 in ad-BMP2 BMSC as compared to the barely detectable levels in ad-GFP BMSC and untransduced BMSC (Fig 2E)
Summary
Bone regeneration using tissue engineering approach, which utilizes mesenchymal stem cells grown in an osteoconductive scaffold and with osteoinductive growth factors is a promising approach and has been studied for over a decade [1]. Use of supraphysiological dose is associated with side effects such as: heterotropic ossification [6], osteolysis [7] and structurally abnormal bone formation [8]. This highlights the need for a more physiological and efficient alternative approach for growth factor delivery. Gene delivery technique, where a therapeutic gene is delivered into a defect site by transgenes employing viral or non-viral vectors, has gained a considerable interest in bone tissue engineering (BTE) [9,10,11]
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