Roles Of Transcription Factors Satb2 And Runx2 In The Differentiation Of Dental Follicle Cells And Bone Marrow Stromal Cells And Their Effects On Regeneration Of Bone And Periodontal Tissues

Periodontal diseases, the most common oral epidemics in adults, destroy tooth supporting alveolar bones and yield tooth loosening and eventual tooth loss. Much attention has been attracted to the regeneration of periodontal tissues but the molecular and cellular bases of periodontal tissue formation, repair and regeneration are still poorly understood despite considerable research effort in this area. According to the classical theory of periodontal development, the dental follicle cells are the origin for periodontal tissues, namely cementum, periodontal ligament (PDL) and alveolar bone. It has been reported that dental follicle cells are plastic adherent and colony forming cells expressing the putative mesenchymal stem cell markers. However, controversies still exist regarding the cellular mechanisms underlying periodontal development. Furthermore, the fact that attempts to regenerate the periodontal tissues have not been successful may be in part due to a lesser degree of understanding of the molecular events leading to the initiation and development of root and periodontal tissues. Based on these facts, it is of great significance to isolate and characterize mesenchymal stem cells existing in the dental follicle as well as investigate the regulating molecular factors involved in the periodontal development. Satb2 is a nuclear matrix protein and expressed in branchial arches and in cells of the osteoblast lineage. Satb2-/- embryos show multiple craniofacial defects that include a significant truncation of the mandible, a shortening of the nasal and maxillary bones, malformations of the hyoid bone, and a cleft palate, that resemble those observed in humans carrying a translocation in Satb2 and defects in osteoblast differentiation and function. It is suggested that in craniofacial reconstruction Satb2 can be a robust osteoinductive molecule recruiting other transcription factors to form a platform or a molecular node for a transcriptional network. It can synergize, amplify and thus exponentially augment the activity of multiple osteogenic factors including Runx2, Osx and Atf4 regulating skeletal development and osteoblast differentiation. However, given the fact that Satb2 is a newly discovered osteogenic transcription factor, its effects in periodontal development have not been reported. Another important osteogenic transcription factor, Cbfa1/Runx2 has been identified as "a master gene" for the differentiation of osteoblasts. Analysis of Runx2-deficient mice has revealed a complete absence of mature osteoblast and ossification with only a few immature osteoblasts which expressed ALP weakly but not OPN and osteocalcin (OCN). Because of the critical role Satb2 and Runx2 play during early development, the corresponding gene knockout (KO) models have resulted in severe phenotypes and immediate death after birth. This has prevented any detailed analysis of the teeth, especially of the periodontal development. Consequently, there is limited direct evidence for the role of these genes and their products in root formation. In this presented study, the pluripotency of mesenchymal stem cells residing in the dental follicle tissues was further evaluated. Furthermore, the roles of Satb2 and Runx2 in osteogenic differentiation and their effects in promoting periodontal regeneration were investigated.Materials and Methods:PartⅠ: A heterozygote BACT-EGFP male mice in which an enhanced green fluorescent protein was driven by a beta-actin promoter and cytomegalovirus enhancer was cross bred with a homozygote mBSP9.0Luc female mouse in which a BSP promoter was linked to a firefly luciferase reporter. The mice with both Luc and GFP genes were named as mBSP9.0Luc/BACT-EGFP. Dental follicle cells were isolated from 5- to 7-day old mBSP9.0Luc/BACT-EGFP mice and were induced to differentiate towards osteoblasts, adipocytes and chondrocytes, respectively. Alizarin red staining, oil red O staining, and alcian blue staining were performed to detect the differentiation. Bone marrow stromal cells isolated from 6- to 8-week-old mBSP9.0Luc/BACT-EGFP mice served as control.PartⅡ: In situ hybridization was performed to investigate the expression pattern of Satb2 in E14.5 mouse embryonic jaws and tooth germs. Satb2 overexpression was achieved by transfecting pcDNA3.1-Satb2 into bone marrow stromal cells and dental follicle cells. Semi-quantitative RT-PCR was performed to determine the expression levels of bone matrix proteins, osteogenic transcription factors and vascular epithelial growth factor A (VEGFA). Satb2 overexpression in murine preosteoblastic MC3T3 cells was also achieved by infection with retroviral construct pBABE-hygro-Satb2. Scratch wound assay was then performed to determine the migration rate changes in these cells. To test whether Satb2 regulates the transcriptional activity of the Osx gene, the full-length Osx promoter construct (-2020/+13) was co-transfected with a Satb2, or Runx2 expression vector, or both into HEK-293 and MC3T3-E1 cells. Luciferase assay was then performed to evaluate the changes in luciferase expression levels. Furthermore, bone marrow stromal cells and dental follicle cells stably transduced with retroviral construct pBABE-hygro-Satb2 were transplanted into the periodontal wound established in B6D2F1 mice. H&E staining, immunohistochemical staining and histomorphometric evaluation were performed to investigate the roles of Satb2 in periodontal regeneration.PartⅢ: Periodontal wound model and femoral wound model were established in Runx2+/- and Runx2+/+ mice. H&E staining and histomorphometric analysis were performed to evaluate the effect of Runx2 haploinsufficiency on bone regeneration. A gene-activated matrix (GAM) method was used to determine the effects of Runx2 in enhancing bone wound healing. Runx2 cDNA was mixed with a biodegradable bovine type I collagen sponge and was inserted into the periodontal window wounds of mice. Control sponges were collagen matrix without Runx2 cDNA. Histological analysis, immunohistochemical staining, and RT-PCR analysis were performed.Results:PartⅠ: After induction by osteogenic medium for 4 weeks and alizarin red staining, deposition of densely stained extracellular matrix was observed in both dental follicle cell cultures and bone marrow stromal cell cultures. The deposition of alizarin red in dental follicle cells was lower than that in bone marrow stromal cells. After adipogenic and chondrogenic induction, the corresponding special staining methods both demonstrated a weaker staining intensity in dental follicle cell cultures when compared with that in bone marrow stromal cell cultures. Semiquantitative RT-PCR analysis also showed that the expression levels of osteoblast-specific ALP, adipocyte-specific LPL, and chondrocyte-specificα1(Ⅱ) procollagen were significantly lower in dental follicle cells than in bone marrow stromal cells after differential induction.PartⅡ: Satb2 was highly expressed in the dental mesenchymal components of incisors but could not be detected in epithelial components of the tooth germs. Moreover, Satb2 was highly expressed in developing palate and mandibular bone matrix and the expression of Satb2 in the edges of developing palatine processes was strong when these two processes were growing towards each other. Satb2 overexpressing BMSC cultures showed increased expression levels of BSP, Runx2, Osx and VEGFA compared with empty vector transfeced cells. We found BMP4 induced the expression of Satb2 and the induction of BSP, Runx2, Osx and VEGFA by BMP4 showed a different pattern compared with the expressing pattern induced by Satb2. We also found that Satb2 overexpression in DFC enhanced the expression of BSP and Runx2. Furthermore, the migration rate was increased in Satb2 ovexpressing MC3T3 cells. Overexpression of Satb2 enhanced the transcriptional activity of the Osx promoter. Forced expression of Cbfa1 also upregulated the full-length Osx promoter activity and Satb2 augmented the Runx2-mediated activation. H&E staining, immunohistochemical staining and histomorphometric analysis demonstrated that Satb2 overexpression in dental follicle cells and bone marrow stromal cells enhanced osteogenic differentiation in these cells and promoted periodontal regeneration.PartⅢ: Compared with wild-type mice, wound healing was dramatically delayed in Runx2+/- mice characterized by the presence of a small amount of bone near the base of the wounds. The bone defects were largely filled with fibrous connective tissues 3 weeks after surgery. Histological analysis and immunohistochemical staining demonstrated that new bone formation in the Runx2-GAM group was increased compared with control group. The new bone formation almost filled the wound defects 14 days after surgery. The collagen sponge matrix did not seem to elicit significant foreign body reaction in either group.Conclusions: In this presented study dental follicle cells were induced to differentiate towards osteoblasts, adipocytes, and chondrocytes in vitro, which further confirmed the existence of pluripotent mesenchymal stem cells in dental follicle tissues. However, after differential induction and special staining it was found that the staining intensity of dental follicle cell cultures was weaker when compared with that of bone marrow stromal cell cultures, which indicated that the pluripotency of dental follicle cells is weaker than bone marrow stromal cells or the stem cell percentage in dental follicle cells is lower than that in bone marrow stromal cells. The distribution of Satb2 expression in jaws and teeth again indicated that Satb2 plays an important role in jaws and tooth development. Satb2 overexpression enhances the expressing levels of bone matrix proteins and osteogenic factors. Furthermore, Satb2 can synergistically upregulate the expression of Osx mediated by Runx2, which demonstrated that by regulating the ratio of other important transcription factors, Satb2 could promote osteogenic differentiation and improve the quality of newly formed bone. Satb2 also increases the expression level of VEGFA, which indicated its potential role in promoting angiogenesis during tissue regeneration. Furthermore, the expression of Satb2 in dental follicle cells of molar tooth germs during root forming stage indicated that Satb2 plays an important role in root formation. The increased migration rate demonstrated that Satb2 plays a role in promoting engraftment of pre-osteoblasts during wound regeneration. In vivo experiments demonstrated that overexpression of Satb2 in dental follicle cells and bone marrow stromal cells enhanced the osteogenic differentiation of these cells and promoted periodontal regeneration. The haploinsufficiency of Runx2 interferes with the process of bone wound healing, while local application of Runx2 cDNA incorporated into a collagen matrix promotes bone tissue regeneration.