Huw F. Thomas

EXPRESSION OF CORE BINDING FACTOR OSF2/CBFA-1 AND BONE SIALOPROTEIN IN TOOTH DEVELOPMENT

The transcription factor Osf2/Cbfa1 is a key regulator of osteogenic differentiation while BSP, a major non-collagenous protein, is a marker of osteoblastic differentiation. To determine the relationship between Osf2/Cbfa1 and the formation of mineralized tissues in tooth development we have studied the temporal expression of Osf2/Cbfa1 and BSP mRNA using in situ hybridization. These studies show that Osf2/Cbfa1 is expressed early in mesenchymal and epithelial tissues destined to form the mineralized tissues of the tooth and periodontal tissues, whereas BSP provides a specific marker for the differentiated cells in each of these tissues. Expression of Osf2/Cbfa1, but not BSP, was observed in the periodontal ligament indicating that expression of Osf2/Cbfa1 is not restricted to mineralizing tissues.

Characterization of dental follicle cells in developing mouse molar.

Dental follicle has been implicated as the origin of alveolar bone, cementum and periodontal ligament, but there is no direct evidence of their cellular lineage. The present pilot study was designed to characterize the phenotype of cultured cells obtained from the dental follicle of neonatal mouse molars. Developing mandibular molars from 6-day-old CD-1 mice were subjected to 1% trypsin in Hanks balanced salt solution. After trypsinization, the dental follicle was enucleated from the tooth germ and separated from the associated epithelial root sheath. Pure dental follicle tissue was cultured in alpha-minimal essential medium containing 10% fetal bovine serum and antibiotics. The nature of the cultured follicle cells was determined in situ by immunocytochemical staining for type I and III collagen, fibronectin, and alkaline phosphatase expression. Earlier phenotypic markers for mineralization such as bone sialoprotein and osteopontin were also examined by in situ hybridization of matched molar tissues. The extracellular matrix proteins (such as type I collagen and fibronectin) were moderately expressed cytochemically. However, type III collagen was strongly stained. Gene expression of bone sialoprotein and osteopontin was detected in sections of mouse molars of similar age. The ALPase activity showed moderate to strong intensity in these primary cultured cells and responded to 1,25(OH)2 vitamin D3 treatment. Cytokeratin stains were not noted in these cells. In conclusion, the 6-day-old dental follicle cells exhibit partial characteristics of a mineralized tissue-forming phenotype even though the expression of osteopontin, type I collagen and fibronectin was low at this stage.

Regulation of Bone Sialoprotein and Osteopontin mRNA Expression by Dexamethasone and 1,25-dihydroxyvitamin D3 in Rat Bone Organ Cultures

Bone sialoprotein (BSP) and osteopontin (OPN) are prominent components of the extracellular matrix of mineralized connective tissues that have been implicated in the formation and remodelling of bone. Although these proteins have similar biochemical properties and are expressed by bone cells during bone formation it has been suggested that they have different functions and that their expression is regulated independently by hormones and cytokines. The precise role of these proteins has, however, yet to be firmly established. Since steroid hormones strongly influence the formation of bone we have analyzed the effects of glucocorticoids and 1,25 dihydroxyvitamin D3 (1,25-(OH)2D3) on the expression of BSP and OPN mRNAs in developing rat bone in vitro using in situ hybridization. In these studies it has been possible to identify the nature and spatial distribution of the cells that respond to these hormones by changes in sialoprotein expression. When cultured in the presence of the synthetic glucocorticoid, dexamethasone (dex), expression of BSP mRNA by hypertrophic cartilage cells in the tibiae and mandible was dramatically increased as were the number of responding cells indicating that glucocorticoids promote differentiation of hypertrophic cartilage cells as well as osteoblasts. Dexamethasone also stimulated a marked (> 5-fold) increase in OPN expression by osteoblasts and cells lining endosteal and periosteal bone surfaces. In contrast to dex, 1,25-(OH)2D3 suppressed BSP expression in osteoblastic cells whereas OPN expression was strongly (> 5-fold) stimulated in all three cultured bone tissues. Histological examination of the tissues showed that cell viability was retained over the culture period. However, in the presence of 1,25-(OH)2D3 considerable resorption of the tissue was evident, with cement and reversal lines being prominent. The increased expression of BSP and OPN by dex is consistent with the stimulation of bone formation by glucocorticoids, whereas the differential effects of 1,25-(OH)2D3 on BSP and OPN may reflect a stimulation of bone remodelling.

Neoplastic odontogenic epithelial cells express bone sialoprotein

Bone sialoprotein (BSP) is synthesized and secreted by bone-, dentine- and cementum-forming cells and has been implicated in de novo bone formation and mineralization. In this study, we used histological sections of odontogenic neoplasms and performed immunohi stochemical and in situ hybridization analyses. In ameloblastoma, BSP mRNA signals were seen in the neoplastic epithelial cells forming nests, strips and islands. BSP deposition was also seen in the stellate reticulum of the tumour masses revealed by immunohistochemistry using human BSP antibodies. In calcifying epithelial odontogenic tumour, the calcified masses demonstrated positive immunoreactivity to the human BSP antibodies, and the hybridization signals for BSP were located in the cells near the calcified particles. In the calcifying odontogenic cyst, strong BSP signals were seen in cells surrounding the characteristic nests of ghost cells, which often calcify subsequently. BSP protein was also found in these cells by immunohistochemistry. The active expression of BSP in the epithelial elements of the odontogenic tumours of adult patients suggests the activation of this matrix protein gene in the neoplastic process, and that BSP may play an important role in tumour formation and differentiation with respect to pathological calcification.

The effect of various fixatives on the extent of the odontoblast process in human dentine.

Unerupted but fully formed human third molar teeth were prepared for ultrastructural examination using three different fixatives; glutaraldehyde, a mixture of glutaraldehyde and p-formaldehyde and a picric acid-formaldehyde solution. These fixatives have reportedly differing rates of penetration of tissue. Odontoblast processes were found to be limited to the inner-third of crown dentine in all teeth, irrespective of the fixative used.

Wound healing and revascularization: A histologic observation of experimental tooth root fracture

We used dogs as an animal model to generate tooth root fracture and to observe the wound-healing process of the fracture. Histologic examination of the specimens revealed that the early reaction of the wound healing was infiltration of inflammatory cells particularly at the coronal part of the fracture, whereas less inflammation but more abundant collagen fibers were seen at the apical part of the fracture (15 and 30 days). Inflammation lasted for more than 90 days and then subsided. At day 180, bone tissue healing was observed. Revascularization of the pulp tissues reached a high level at the same stage that bone healing took place. Our data suggest that in tooth root fracture, the regeneration of blood vessels is important in the wound-healing process and the revascularization is synchronized with the fracture wound healing. In this animal model the complete hard tissue healing could take as long as 6 months.

Osteocalcin expression in young and aged dental pulps as determined by RT-PCR

Dental pulps were obtained from third molars of young adults (17-25 yr) or from molar teeth of individuals > 50 yr of age and examined for the expression of osteocalcin (OC) mRNA by RT-PCR. OC was selected as a determinant of pulp vitality, because it has long been associated with the production of hard tissue matrix in teeth and bone. For comparative purposes, the expression of OC in each pulp was normalized relative to its housekeeping gene-product GAPDH by the establishment of a OC/GAPDH ratio. This study demonstrated that OC expression, presumably by cells of odontoblast lineage, does not diminish relative to the extant cell population. Our findings suggest, despite a reduction in volume and cell numbers, that the pulps of aging teeth retain a capacity for dentin deposition and a potential for caries and trauma resistance.

Identification of Regulatory Elements Necessary for the Expression of the COL1A1 Promoter in Murine Odontoblasts

Recent studies have indicated that odontoblasts and osteoblasts have unique regulatory mechanisms that control COL1A1 gene expression. We are currently examining the regulation of COL1A1 gene expression in odontoblasts and have produced transgenic mice containing various collagen promoter constructs fused to the indicator gene, chloramphenicol acetyl transferase (CAT). Mandibular first molars were removed from jaws of transgenic mice. Some teeth were assayed for CAT activity (CAT diffusion assays), others were fixed and prepared for immunohistochemistry (CAT antibodies). Our results indicate the CAT activity was present in tooth germs containing promoter constructs longer than 1.719 kb. Immunoreactivity to CAT was confined to the odontoblast cell layer. No CAT activity was present in tooth germs containing a 1.670 kb construct. These data suggest that there are important regulatory elements located between -1.719 kb and -1.670 kb on the collagen promoter in odontoblasts. Examination of sequences in this region of the promoter demonstrates consensus with those known to be involved with binding of translation products of homeobox genes.

DEXAMETHASONE STIMULATES LUCIFERASE GENE EXPRESSION THROUGH THE RAT BONE SIALOPROTEIN GENE PROMOTER IN TRANSGENIC MICE

Bone sialoprotein (BSP) is expressed by differentiated osteoblasts during the initial formation and mineralization of bone matrix. Studies using transgenic mice harboring 2.7 kb of the rat BSP promoter linked to a luciferase reporter gene have shown luciferase activity in bone and other mineralized tissues while most soft tissues tested expressed a much lower level of the reporter gene. To study regulation of the transgene, mice were administered dexamethasone (dex) by intramuscular injection. After 4 h and 24 h, various tissues were dissected from the treated mice as well as from untreated transgenic littermates. Luciferase assays showed that dex stimulated expression of the transgene significantly. In bone tissues, dex increased the average luciferase activity 1.6- to 11-fold compared with control tissues from untreated transgenic mice. The luciferase activity in lung, liver and kidney remained at a low level and showed no increase with dex treatment. In some animals, however, the luciferase activity in brain and skin was also increased after dex administration. These experiments indicate that a transgene comprising 2.7 kb of the rat BSP promoter linked to a luciferase reporter is regulated in a tissue and developmental stage-dependent manner and that glucocorticoid-induced stimulation of BSP gene expression may be mediated within this region of the promoter.