Osterix overexpression in mesenchymal stem cells stimulates healing of critical-sized defects in murine calvarial bone

Abstract

Osterix (Osx) is a zinc-finger-containing transcription factor that is expressed in osteoblasts of all endochondral and membranous bones. In Osx null mice, osteoblast differentiation is impaired, and bone formation is absent. We hypothesized that overexpression of Osx in bone marrow-derived mesenchymal stem cells (BMSCs) would enhance osteogenic differentiation during bone regeneration in vivo. Overexpression of Osx in mouse BMSCs was achieved using retroviral infection together with a green fluorescent protein (GFP) vector to monitor transduction efficiency and determine the source of regenerative cells in implantation studies. Bone regeneration in vivo was evaluated by implanting BMSCs overexpressing Osx into 4-mm calvarial bone defects in adult mice using type I collagen sponge as a carrier. New bone formation in the defects was quantified using radiological and histological procedures 5 weeks after implantation. The results showed that implantation of Osx-transduced BMSCs resulted in 85% healing of calvarial bone defects as detected using radiological analyses. Histological examination of the implants demonstrated that the Osx-transduced group exhibited amounts of newly formed bone that was five times as high as in a group transduced with the empty vector. Immunohistochemistry for GFP showed positive immunoreaction localized to areas of newly engineered bone in the Osx-transduced group. Immunohistochemistry with antibodies against the extracellular matrix protein bone sialoprotein resulted in strong staining in areas of new bone formation. In addition, the clonal BMSCs showed an osteogenic potential similar to that of primary cultures of BMSCs, suggesting the usefulness of this model in bone tissue engineering. These results indicate that ex vivo gene therapy of Osx is a useful therapeutic approach in regenerating adult bone tissue.

FIG. 1

Schematic drawing of replication-competent, subgroup-A avian leukosis viral vector (RCAS)/avian retroviral receptor (TVA) system. A viral vector-carrying gene of interest (e.g., osterix (Osx)) will specifically target bone marrow–derived mesenchymal stem cells engineered to express TVA receptor. This infection does not cause viral spreading or immunity, because gag (for a structural protein), pol (for a viral enzyme), or env (for an envelope glycoprotein) are poorly expressed in mammals. Long terminal repeats (LTRs) serve as a constitutive promoter, resulting in expression of the Osx gene. CMV, cytomegalvirus; RNA, ribonucleic acid.

FIG. 2

The replication-competent, subgroup-A avian leukosis viral vector (RCAS)/avian retroviral receptor (TVA) retroviral system induces high levels of expression of osterix (Osx) in bone marrow–derived mesenchymal stem cells (BMSCs) cultured in maintenance media. RCAS-Osx-transduced BMSCs and empty vector-transduced BMSCs were cultured in maintenance media (non-differentiating) for different times. (A) Representative image of semiquantitative reverse transcriptase polymerase chain reaction analyses of Osx expression. (B) Levels of Osx were normalized with those of the loading control glyceraldehyde-3-phosphate dehydrogenase in 3 independent experiments. ^ap <0.05 vs. 1-week time point.

FIG. 3

Osterix (Osx) promotes bone healing in the skull assay. (A) Representative images of x-ray radiograph of calvarial defects 5 weeks after bone marrow–derived mesenchymal stem cell (BMSC) implantation. Defect treatments, from left to right: group 1, unfilled defect; group 2, collagen alone; group 3, collagen + primary BMSCs; group 4, collagen + clonal BMSCs; group 5, collagen + RCAS vector-transduced BMSCs; group 6, collagen + RCAS-Osx-transduced BMSCs. (B) Percentage of healing. The extent of mineralization filling the calvarial defects was monitored using densitometry 5 weeks after implantation, and then the percentage of healing was calculated in all groups as described in the Materials and Methods section. ^a p <0.05 vs. group 1 or group 2; ^b p <0.05 vs. group 3, 4, and 5.

FIG. 4

Histological analyses of implanted calvarial bone defects. (A) Calvarial specimens were stained with hematoxylin and eosin. 1. Group 1, unfilled defect; 2. group 2, collagen alone; 3. group 3, collagen + primary bone marrow–derived mesenchymal stem cells (BMSCs); 4. group 4, collagen + clonal BMSCs; 5. group 5, collagen + RCAS vector-transduced BMSCs; 6. group 6, collagen + RCAS-OSX-transduced BMSCs. Photographs were taken at 40×. The arrowheads designate the surgical margins. (B) Areas of new bone formation were calculated as percentages with respect to total area and showed underneath representative pictures. ^a p <0.05 vs. group 1 or group 2; ^bp <0.05 vs. group 3, 4, and 5.

FIG. 5

Immunohistochemical staining for bone sialoprotein (BSP; original magnification, 200×) and immunolocalization of green fluorescent protein (GFP) expression (original magnification, 400×) within newly formed bone areas. (A) Expression of BSP in the sections was detected using immunohistochemical staining using a BSP monoclonal antibody (1:25; Chemicon International, Temecula, CA) followed by counterstaining with hematoxylin. Calvarial specimens were from the following groups: 1. unfilled defect; 2. collagen alone; 3 collagen + primary bone marrow–derived mesenchymal stem cells (BMSCs); 4. collagen + clonal BMSCs; 5. collagen + RACS vector-transduced BMSCs; 6. collagen + RCAS-OSX-transduced BMSCs. BSP expression (reddish staining) is localized to osteoblasts and osteocytes within newly formed bone. (B) Immunohistochemistry for GFP expression was performed to analyze the fate of the implanted BMSCs within areas of newly formed bone. Counterstaining with hematoxylin was performed to detect the nuclei of the cells in the newly formed bone areas. Negative staining was found in host bone (HB, 1) and clonal BMSC implant without GFP viral infection (2). GFP expression was detected in the specimens from defects filled with collagen + empty RCAS-transduced BMSCs (3), and defects filled with collagen + RCAS-OSX-transduced BMSCs (4), which were labeled with GFP viral co-infection. Signals for GFP staining were localized in osteoblasts and osteocytes within newly formed bone (NB), periosteum, and fibrous connective tissue (FT) surrounding new bone. BM, bone marrow. Color images available online at www.liebertpub.com/ten.