Lentiviral-Mediated Integrin α5 Expression in Human Adult Mesenchymal Stromal Cells Promotes Bone Repair in Mouse Cranial and Long-Bone Defects

Oral and Maxillofacial Surgery Department, Carmel Medical Center , 32000 Haifa, Israel.
Human gene therapy (Impact Factor: 3.76). 09/2011; 23(2):167-72. DOI: 10.1089/hum.2011.059
Source: PubMed


Abstract Adult human mesenchymal stromal cells (hMSCs) are an important source for tissue repair in regenerative medicine. Notably, targeted gene therapy in hMSCs to promote osteogenic differentiation may help in the development of novel therapeutic approaches for bone repair. We recently showed that α5 integrin (ITGA5) promotes osteoblast differentiation in bone marrow-derived hMSCs. Here, we determined whether lentiviral (LV)-mediated expression of ITGA5 in hMSCs derived from the bone-marrow stroma of healthy individuals may promote bone repair in vivo in two relevant critical-size bone defects in the mouse. In a first series of experiments, control or LV-ITGA5-transduced hMSCs were seeded on collagen-based gelatin sponge and transplanted in a cranial critical-size defect (5 mm) in Nude-Foxn1nu mice. Microcomputed tomography and quantitative histological analyses after 8 weeks showed no or little de novo bone formation in defects implanted with collagen sponge alone or with hMSCs, respectively. In contrast, implantation of collagen sponge with LV-ITGA5-transduced hMSCs showed greater bone formation compared with control hMSCs. We also tested the bone-repair potential of LV-mediated ITGA5 expression in hMSCs in a critical-size long-bone defect (2 mm) in femur in Nude-Foxn1nu mice. Bone remnants were stabilized with external fixation, and control or LV-ITGA5-transduced hMSCs mixed with coral/hydroxyapatite particles were transplanted into the critical-size long-bone defect. Histological analysis after 8 weeks showed that LV-ITGA5-transduced hMSCs implanted with particles induced 85% bone regeneration and repair. These results demonstrate that repair of critical-size mouse cranial and long-bone defects can be induced using LV-mediated ITGA5 gene expression in hMSCs, which provides a novel gene therapy for bone regeneration.

Download full-text


Available from: Pascal Vaudin,
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Promoting osteoblastogenesis remains a major challenge in disorders characterized by defective bone formation. We recently showed that the alpha 5 integrin subunit (ITGA5) is critically involved in human mesenchymal cell osteoblast differentiation. In this study, we determined the potential of pharmacological ITGA5 activation by a synthetic cyclic peptide (GA-CRRETAWAC-GA) on murine osteoblast differentiation and function in vitro and bone formation in vivo. Peptide-mediated activation of ITGA5 in murine C3H10T1/2 mesenchymal cells resulted in the generation of the integrin-mediated cell signals FAK and ERK1/2-MAPKs. In vitro, peptide-based activation of ITGA5 protected from cell apoptosis but did not affect cell adhesion or replication, while it enhanced the expression of the osteoblast marker genes Runx2 and type I collagen and increased extracellular matrix (ECM) mineralization as also found with bone morphogenetic protein-2 (BMP2), a standard bone anabolic factor. When injected on adult mouse cranial bone for 3 weeks, the peptide-mediated activation of ITGA5 increased bone thickness by twofold, an effect also induced by BMP2. Histomorphometric analysis showed that this anabolic effect resulted from decreased cell apoptosis and increased bone forming surfaces and bone formation rate (BFR). We conclude that pharmacological activation of ITGA5 in mesenchymal cells is effective in promoting de novo bone formation as a result of increased osteoprogenitor cell differentiation into osteoblasts and increased cell protection from apoptosis. This peptide-based approach could be used therapeutically to promote the osteogenic capacity of osteoblast progenitor cells and to induce de novo bone formation in conditions where osteoblastogenesis is compromised.
    Journal of Cellular Biochemistry 09/2012; 113(9):3029-38. DOI:10.1002/jcb.24181 · 3.26 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: Background: Genetic modification of human bone marrow stem cells (hBMSCs) before administration to a patient is emerging as a viable approach to creating tailored cells that perform effectively in a clinical setting. To this end, safe delivery systems are needed that can package therapeutic genes into nanoparticles for cellular delivery. Methods: We evaluated different plasmids on gene expression and compared the effective plasmids directly in hBMSCs. Then, we evaluated the transfection efficiencies of the polymeric carriers linoleic acid-substituted polyethylenimine (PEI-LA), polyethylenimine (PEI)-25, and PEI-2 using flow cytometry. We used 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide to compare the toxicity of PEI-LA and PEI-25 on hBMSCs. We further assessed bone morphogenetic protein-2 (BMP-2) secretion and the osteogenic activity of hBMSCs transfected with the polymeric (PEI-LA and PEI-25) gWIZ-BMP-2 complex. Results: Unlike the transformed cells that gave robust (>50%) transfection, only a few percent (<10%) of hBMSCs was transfected by the developed nanoparticles in culture. The plasmid DNA design was critical for expression of the transgene product, with the choice of the right promoter clearly enhancing the efficiency of transgene expression. Using the in-house designed PEI-LA, hBMSCs secreted BMP-2 in culture (~4 ng BMP-2/10(6) cells/d), which indicates the feasibility of using PEI-LA as a delivery system. Furthermore, we demonstrated an increased osteogenic activity in vitro for hBMSCs transfected with the PEI-LA containing the BMP-2 expression system. Conclusions: These results provide encouraging evidence for the potential use of a low toxic PEI-LA to genetically modify hBMSC.
    Journal of Surgical Research 12/2012; 183(1). DOI:10.1016/j.jss.2012.11.061 · 1.94 Impact Factor
  • [Show abstract] [Hide abstract]
    ABSTRACT: The ageing skeleton experiences a progressive decline in the rate of bone formation, which can eventually result in osteoporosis-a common disease characterized by reduced bone mass and altered bone microarchitecture which can result in fractures. One emerging therapy involves the identification of molecules that target bone-marrow mesenchymal stromal cells (MSCs) and promote their differentiation into osteoblasts, thereby counteracting bone loss. This Review highlights the discovery that some integrins, a family of heterodimeric transmembrane proteins that can interact with matrix proteins and generate intracellular signals, can be targeted to promote homing of MSCs to bone, osteogenic differentiation and bone formation. Specifically, priming of the α(5)β(1) integrin, which is required for osteoblastic differentiation of MSCs, leads to increased bone formation and improved bone repair in mice. Additionally, treatment with a peptidomimetic ligand of the α(4)β(1) integrin coupled to an agent with a high affinity for bone improves the homing of MSCs to bone and promotes osteoblast differentiation and bone formation, leading to increased bone mass in osteopenic mice. Strategies that target key integrins expressed by MSCs might, therefore, translate into improved therapies for age-related bone loss and possibly other disorders.
    Nature Reviews Endocrinology 01/2013; 9(5). DOI:10.1038/nrendo.2013.4 · 13.28 Impact Factor
Show more