Abstract
Calcitonin gene-related peptide (CGRP) promotes osteoblast recruitment and osteogenic activity. However, no evidence suggests that CGRP could affect the differentiation of stem cells toward osteoblasts. In this study, we genetically modified adipose-derived stem cells (ADSCs) by introducing the CGRP gene through adenoviral vector transduction and investigated on cellular proliferation and osteoblast differentiation in vitro and osteogenesis in vivo as well. For the in vitro analyses, rat ADSCs were transducted with adenoviral vectors containing the CGRP gene (Ad-CGRP) and were cultured in complete osteoblastic medium. The morphology, proliferative capacity, and formation of localized regions of mineralization in the cells were evaluated. The expression of alkaline phosphatase (ALP) and special markers of osteoblasts, such as Collagen I, Osteocalcin (BPG) and Osteopontin (OPN), were measured by cytochemistry, MMT, RT-PCR, and Western blot. For the in vivo analyses, the Ad-CGRP-ADSCs/Beta-tricalcium phosphate (β-TCP) constructs were implanted in rat radial bone defects for 12 weeks. Radiography and histomorphology evaluations were carried out on 4 weeks and 12 weeks. Our analyses indicated that heterogeneous spindle-shaped cells and localized regions of mineralization were formed in the CGRP-transduced ADSCs (the transduced group). A higher level of cellular proliferation, a high expression level of ALP on days 7 and 14 (p<0.05), and increased expression levels of Collagen I, BPG and OPN presented in transduced group (p<0.05). The efficiency of new bone formation was dramatically enhanced in vivo in Ad-CGRP-ADSCs/β-TCP group but not in β-TCP group and ADSCs/β-TCP group. Our results reveal that ADSCs transduced with an Ad-CGRP vector have stronger potential to differentiate into osteoblasts in vitro and are able to regenerate a promising new tissue engineering bone in vivo. Our findings suggest that CGRP-transduced ADSCs may serve as seed cells for bone tissue engineering and provide a potential way for treating bone defects.
Highlights
Bone is a highly vascularized and innervated connective tissue that is subject to continuous remodeling and renovation
The Calcitonin gene-related peptide (CGRP)-adipose-derived stem cells (ADSCs) appeared as heterogeneous population of spindle-shaped cells; despite some colony growth mode, while the growth was evenly distributed, long spindleshaped cells dominated
Modified bone tissue engineering is an attractive approach with great potential for repairing bone defects that result from trauma, surgical resection, and congenital deformity corrections [3,4,5]
Summary
Bone is a highly vascularized and innervated connective tissue that is subject to continuous remodeling and renovation. Many complex clinical conditions require bone regeneration in large quantities, such as large bone defects, atrophic nonunions and osteoporosis [1]. Bone tissue engineering is a promising treatment for these diseases [2]. Genetically-modified, engineered bone tissue is an attractive approach with great potential for repairing large bone defects or nonunions [3,4,5]. Mesenchymal stem cells (MSC) hold great promise for future translational research and clinical applications in many fields. Much research has focused on bone mesenchymal stem cell (BMSCs) isolated from bone marrow; harvesting bone marrow causes considerable discomfort to the patient and yields a relatively small number of cells. Animal and clinical studies have shown that ADSCs are capable of repairing damaged skeletal tissue or largebone segmental defects [9,10]
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