During primary dentin formation, differentiating primary odontoblasts secrete an organic matrix, consisting principally of type I collagen and non-collagenous proteins, that is capable of mineralizing at its distal front. In contrast to ameloblasts that form enamel and undergo programmed cell death, primary odontoblasts remain metabolically active in a functional tooth. When dentin is exposed to caries or by operative procedures, and when exposed dentinal tubules are treated with therapeutic dental materials, the original population of odontoblasts is often injured and destroyed. The characteristics of the replacement pool of cells that form reparative dentin and the biologic mechanisms that modulate the formation of this matrix are poorly understood. Based on the hypothesis that events governing primary dentinogenesis are reiterated during dentin repair, the present study was designed to test whether cells that form reparative dentin are odontoblast-like. Cervical cavities were prepared in rat first molars to generate reparative dentin, and animals were killed at various time intervals. In situ hybridization with gene-specific riboprobes for collagen types I and III was used to study de novo synthesis by cells at the injured dentin-pulp interface. Polyclonal antibodies raised against dentin sialoprotein (DSP), a dentin-specific protein that marks the odontoblast phenotype, were used in immunohistochemical experiments. Data from our temporal and spatial analyses indicated that cells forming reparative dentin synthesize type I but not type III collagen and are immunopositive for DSP. Our results suggest that cells that form reparative dentin are odontoblast-like.

The TUNEL (transferase-mediated, dUTP-biotin nick end labeling) method for in situ labeling of DNA strands was utilized to localize DNA fragmentation in cells involved in tooth formation in the neonatal mouse and hamster. Positive reactions for the presence of DNA fragments were obtained in some epithelial cells of the cervical loop region of incisors, late secretory, transitional and early maturation stage ameloblasts, stratum intermedium cells and in shortened ameloblasts just before eruption. Also, cells of the periodontal ligament of the continuously erupting incisors stained positive shortly before eruption. Odontoblasts were negative but became strongly positive during the formation of physiological osteodentin at the tip of developing incisors. Osteodentin matrix and the surfaces of unerupted enamel and cementum just prior to eruption stained for DNA fragments as well. DNA fragmentation could be elicited in odontoblasts and underlying pulpal tissues of mature erupted molars after mechanical injury to the odontoblast processes during cavity preparation. We conclude that, in rodents, DNA fragmentation and cell death are biological processes which take place in a variety of cells involved in formation of teeth. The TUNEL staining technique is a simple but powerful tool to examine the fate of cells and tissues undergoing either programmed cell death (apoptosis) or fragmentation of nuclear DNA induced by external factors leading to pathological changes.

While transforming growth factor-beta 1 (TGF-beta 1) and its related mammalian isoforms TGF-beta 2 and TGF-beta 3 are coexpressed in developing tooth organs, the specific biological role of each isoform is unknown. To delineate the role of TGF-beta 1 in odontogenesis, we have studied tissues from mice that lack a functional TGF-beta 1 gene. Histologic analyses revealed that in TGF-beta 1 (-/-) mice, tooth morphogenesis, cytodifferentiation and histogenesis were unaffected. Using in situ hybridization we studied the patterns of distribution of TGFs-beta 1, beta 2 and beta 3 in the TGF-beta 1 (+/+, +/- and -/-) genotypes. Our results indicate no detectable TGF-beta 1 mRNA in null tissues while TGFs-beta 2 and beta 3 showed normal temporal-spatial patterns of distribution. Using antibodies against TGF-beta 1, we observed immunoreactive TGF-beta 1 in tissues from null mice suggesting that maternally-derived TGF-beta 1 may be involved in the rescue of several developmental events in TGF-beta 1 knockout mice.

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