Transgenic stem cells for craniofacial bone reconstruction

Abstract

Bone loss from the cranio-maxillofacial region is a major clinical problem affecting patients worldwide. Conventional treatment strategy includes the use of autogenous or allogeneic bone, biomaterials, and osteogenic growth factors. However, there has been no effective therapy for most cases so far. Stem cell-based gene therapy is the latest research method with possible applications in humans. The present study aims to (1) characterize rabbit mesenchymal stem cells (MSCs) relating to growth pattern, surface antigens, and the potential for multi-differentiation; (2) determine the transduction efficiency and duration of recombinant adeno-associated virus2 carrying enhanced green fluorescent protein (rAAV2EGFP) reporter gene in rabbit MSCs and study the effects of rAAV2EGFP transduction on stem cells’ phenotype and capacity of multi-differentiation; (3) evaluate the differentiation characteristics of rabbit MSCs following recombinant adeno-associated virus 2 carrying bone morphogenetic protein 2 gene (rAAV2BMP2) transduction; (4) investigate whether MSCs transduced by rAAV2BMP2 could successfully induce bone regeneration in rabbit critical-size cranial defects.

MSCs were isolated from bone marrows of rabbit tibias and cultured. Cell counting and colony-forming assays demonstrated that growth rates of MSCs dropped substantially with increasing passages. Flow cytometry on MSCs at passage 1 showed that cells expressed high level of CD49a and low level of CD44 as well as stage-specific embryonic antigen 4 (SSEA4). Multi-differentiation and reverse transcriptase-polymerase chain reaction (RTPCR) tests demonstrated that rabbit MSCs were capable to differentiate into osteocytes, chondrocytes and adipocytes. Immunofluorescence microscopy showed that rabbit MSCs produced a series of hematopoietic growth factors, including stem cell factor (SCF), vascular endothelial growth factor-A (VEGFA) and granulocyte macrophage colony-stimulating factor (GMCSF).

Subsequently, rabbit MSCs were transduced with rAAV2EGFP in vitro. By comparing the transduction efficiency with different doses of rAAV2EGFP particles, multiplicity of infection (MOI) of 1 x 10 4 was identified as an optimal parameter for the transduction of rAAV2 in rabbit MSCs. Fluorescent microscopy demonstrated long-term expression of EGFP in rabbit MSCs after transduction both in vitro and in vivo. In addition, cell proliferation assay, adipogenic induction test and flow cytometry showed that rAAV2EGFP transduced MSCs exhibited a similar pattern with non-transduced cells on the cell growth, capacity of adipogenic differentiation and expression of surface antigens, indicating that rabbit MSCs maintain their stem cell properties after rAAV2EGFP transduction.