Molecular Regulation of Bone Formation
Our group is interested in several areas related to the molecular control of bone formation. We use a variety of genetic, genomic, and transgenic mouse approaches to study the signaling pathways involved and the mechanisms by which they are regulated.
Wnt Signaling and Bone Formation
Our laboratory has provided strong evidence that microRNAs (miRNAs) play a strong role in regulating Wnt signaling and osteogenic differentiation by controlling the expression levels of DKK1. We performed a miRNA microarray analysis and identified specific miRNAs that target a specific binding site on the DKK1 protein 3’UTR to down-regulate the DKK1 protein level (Fig. 1).
Figure 1. Changes in miRNA expression profiles as indicated by miRNA microarray analysis. Original images of the chips. The chip detects miRNA transcripts listed in Sanger miRBase Release 11.0; B) Data analysis revealed groups of miRNAs which were down- or up- regulated during osteogenic differentiation.
We then determined through in situ hybridization, that the miRNA is highly expressed in osteoblasts during bone development (Fig. 2), indicating that regulatory effect of miRNAs is mandatory in the initiation and progression of osteoblastic differentiation.
Figure 2. In situ hybridization showed that miR-335-5p is highly expressed in osteoblasts during bone development. At E13.5, miR-335-5p was highly expressed in osteoblasts surrounding the developing mandible and palatine. However in chondrocytes, the expression of miR-335-5p was barely detected. At E16.5, the staining intensity for miR-335-5p was even stronger in osteoblasts.
This effect may be essential in manipulating bone formation. Further studies investigating the role miRNAs plays in the regulation of ostegenic differentiation will shed light and possibly provide valuable insight in therapeutic treatments for bone loss diseases.
Stem Cells & Bone Tissue Engineering to Repair Craniofacial Defects
We recently investigated the capabilities and advantages of periodontal regeneration using induced pluripotent stem (iPS) cells and enamel matrix derivatives (EMD). Using several approaches we have been able to generate embryoid bodies from human and murine iPS (Fig. 3).
Figure 3. Morphology of embryoid bodies (EB) formed from human and mouse iPS cells. A) free-floating EB formed from human iPS cells after 5 days of suspension culture in non-adherent culture dishes; B) mouse EB formed from mouse iPS cells by drop-suspension (40×).
We have demonstrated the effect of EMD gel on iPS cells in vitro, and then performed tissue engineering techniques with iPS cells and EMD in periodontal defects. We discovered that transplantation of mouse iPS cells combined with EMD, repairs mouse periodontal defects by prompting the formation of cementum, alveolar bone, and normal periodontal ligament (Fig. 4). At 24 days post-surgery, the experimental group (Fig 4B) showed significantly more new alveolar bone formation, new cementum covering almost the whole denuded surface, and regenerated PDL separated the new bone from new cementum than the control group (Fig 4A), which showed inadequate alveolar bone reconstruction, insufficient cementum regeneration, and new PDL separated the new bone from new cementum.
Figure 4. Histomorphometriclogical analysis showed transplantation of iPS cells combined with EMD enhanced the whole periodontal tissues regeneration. A) control group, silk scaffold + EMD; B) experimental group, silk scaffold + EMD + iPS cells; Line: suppositional margins of defect; nAB: new alveolar bone; nPL: new periodontal ligament; arrow: new cementum. Photographs were taken at 100X.
Control of Bone Metastases
We determine the role of bone sialoprotein (BSP) overexpression in osteolytic metastasis, using two homozygous transgenic mouse lines: CMV-BSP mice (where BSP is elevated in all tissues) and Ctpsk-BSP mice (where BSP is only elevated osteoclasts). CMV-BSP mice and CtpsK-BSP mice received intracardiac injections of breast cancer cells labeled by luciferase reporter. The IVIS imaging showed that skeletal and systemic metastasis of breast cancer cells were dramatically increased in CMV-BSP mice, which is a result of BSP over expression in all cell types. In CtspK-BSP mice the tumor migration was limited to bone, and was larger than that in the wild type, due to the restricted BSP overexpression in osteoclasts. The CtspK-BSP mice received intratibial injections of breast cancer cells labeled by luciferase, to demonstrate the increased osteoclastogenesis and osteolytic activities (Fig. 5). We were able to establish that osteoclasts differentiate via the interaction with secreted proteins and host tissue-derived BSP also plays an important role in breast cancer bone metastasis.
Figure 5. Radiographs of the right hindlimbs from wild type (WT) and CtpsK-BSP (TG) mice 3 weeks after intratibial inoculation showing increased osteoclastogenesis and osteolysis.
Approaches to Osteointegration of Implants
Our laboratory transplanted double-labeled bone marrow stromal Cells (BMSCs) into nude mice through an intracardiac injection, 5 weeks after injection, Osx retrovirus was applied to the femur and titanium implants were inserted. Immunohistochemical staining was performed on implantation site 7 days after surgery to assess the newely formed bone area (Fig 6). This demonstrated that BMSCs can recruit from peripheral circulation and participate in wound healing and osseointegration after implantation.
Figure 6. Immunohistochemical staining 7 days after transplantion of BMSCs, expression levels of luciferase, BSP, and GFP were examined.
MicroCT analysis demonstrated that 14 days after implantation, the implants were successfully integrated with the host bone tissue in both the Osx group and control group (Fig 7).
Figure 7. MicroCT analysis 14 days after implantation demonstrating integration of the implants.
The newly formed bone density surrounding the implant was higher in the Osx group than in the control group. We found that the participation of BMSCs in the osseointegration after implantation, and the over expression of Osx accelerates osseointegration.