Tooth development is a precisely regulated process. During its early stages, epithelial–mesenchymal interactions result in the timed and intimately sequenced formation of coronal enamel and dentine. At this early stage, the mesenchymal cells and matrix comprising the surrounding dental follicle lack organization and pattern. However, as root formation is initiated and progresses, the amorphous character of the dental follicle is gradually replaced by the structured morphology of the periodontal ligament. The ligament is a unique tissue in terms of composition, location, and function. It maintains a constant soft-tissue space between the mineralization fronts of root cementum and alveolar bone while providing a functional and dynamic fibrous link between these two tissues. The mechanisms which promote the genesis of the periodontal ligament while inhibiting ankylosis of bone to root remain largely unknown. It has been suggested that specific signals, including mechanical stimuli and extracellular factors/proteins, play a part in regulating the behaviour of the periodontal ligament, and it is probable that these signals are not only operational during the development of the ligament but also during periods of its maintenance, repair and regeneration.

One logical candidate for a role in controlling the activity of the periodontal ligament is type XII collagen, a fibril-associated collagen found in high concentrations in ligamentous cells and tissues. Type XII collagen belongs to a subclass of the collagen superfamily termed fibril-associated collagens with interrupted triple helices, or FACIT. Collagens in this class include types IX, XII, XIV and XVI, and are similar in having short triple-helical domains separated by non-triple-helical regions. The triple-helical regions have an affinity for fibrillar collagen; it is believed that the non-triple-helical domains provide sites for interaction with other extracellular matrix molecules. Type XII collagen consists of three 1 (XII) chains where two domains are triple helical and three are non-triple helical. Like other FACIT molecules, type XII collagen binds to collagen fibrils through its triple-helical domains and to collagen fibrils as well as other extracellular matrix molecules through its non-helical domains.

Type XII collagen was first reported in periodontal ligament by Yamauchi et al. (1986). A close association of type XII collagen with type I collagen fibrils is suggested, based on common areas of location, i.e., dense connective tissues such as tendon, ligament, perichondrium and periosteum as found in both chicken and mouse. This association prompted several researchers to investigate the functional relation of type XII to type I collagen. Nishiyama et al. (1994) demonstrated that type XII collagen promotes fibroblast-mediated contraction of type I collagen gel and suggested that this results from increased mobility of hydrated collagen fibrils within the gel. These findings also suggest that type XII collagen may have an important role in modulating the biomechanical properties of collagenous tissues.

It is particularly interesting that type XII collagen is found in relatively high concentrations in ligamentous tissues, including the periodontal ligament. Using periodontal tissues obtained from 25- and 40-day-old Sprague–Dawley rats, Karimbux et al. (1992) demonstrated the expression of type XII collagen at two distinct developmental time-points in rat periodontal ligament. Importantly, expression was more intense at day 40, where teeth are fully functioning, than at day 25, where root formation has not yet been completed. In contrast, the expression of type I collagen (2(I)) decreased with maturity. Karimbux concluded, based on the temporal and spatial expression of these collagens, that the expression of type XII collagen may be important to the structural alterations that occur within the periodontal ligament during development, as required to maintain a mature ligament that can withstand the forces of occlusion.

We here consider the hypothesis that the expression of type XII collagen is temporally linked with root formation and with the development and maturation of the periodontal ligament. We predicted that the expression of type XII collagen would increase at later stages of root development as the fibres of the ligament insert and align to the root. Using the developing mouse molar root as the experimental tissue and in situ hybridization as the investigational tool, expression of type XII collagen was compared to that of type I collagen, the most abundant collagen in periodontal tissues, at sequential time-points during the formation of the root/periodontal ligament.

All procedures were approved by the University of Michigan Committee on the Use and Care of Animals and were in compliance with state and federal laws. Neonatal offspring (n=21) from timed-pregnant female CD-1 mice (Charles River Laboratories, Cambridge, MA) were killed by decapitation at developmental days 21, 24, 27, 33, 41, 60, and at later times (i.e., adult mice). In total, mandibular tissue was isolated from 21 animals (three animals per time-point). [For clarification, the vaginal plug date was designated as day 0. Thus developmental day 21 by the notation used here relates to neonatal day 3, i.e., mice are usually born at day 19 gestation.]

Mandibles were dissected from surrounding tissues and then hemisected by incision through the symphysis. Tissues were immersed immediately in Bouin’s fixative (0.9% picric acid, 9% vol/vol formaldehyde, and 5% acetic acid; Polysciences, Warrington, PA). Tissues at or beyond day 27 of development were demineralized in acetic acid and formal saline (4% formaldehyde in 0.85% NaCl+10% acetic acid) until an acceptable radiographic end-point was achieved (1–5 days). All tissues were hydrated through a graded series of ethanols, cleared in xylenes, and embedded in low melting-point paraffin (Tissue Prep; Fisher Scientific, Fair Lawn, NJ). Serial 7-?m sections were cut in a coronal plane and placed on 3-aminopropyltriethoxysilane-coated (Sigma No. A1435; Sigma, St. Louis, MO) slides. Tissue sections from five individual animals were analysed at each developmental time-point.

The probes used were type I collagen [bovine (I)], a gift from Drs Marion Young and Larry Fisher (NIDR, NIH) and type XII collagen [mouse 1(XII)], a gift from Drs Bjorn Olsen and Suk Paul Oh (Harvard Medical School). This type XII collagen probe encodes the carboxyl region of the non-collagenous domain of mouse type XII collagen and recognizes both the short and long spliced variants of type XII collagen. Type I collagen probe was linearized with XbaI and transcribed with T7 RNA polymerase (sense), or linearized with XhoI and transcribed with T3 RNA polymerase (antisense). Type XII collagen probe was linearized with BamHI and transcribed with T7 RNA polymerase (sense) or linearized with XhoI and transcribed with T3 RNA polymerase (antisense).

After linearization the type I collagen cDNA probes were transcribed and labelled with Amersham in situ grade [³⁵S] uridine triphosphate and Riboprobe Gemini SystemR (Promega Biotec, Madison, WI). Linearized type XII collagen cDNA probes were transcribed and labelled with both [³⁵S] uridine triphosphate and [³⁵S] cytidine triphosphate from Amersham using a Maxi-Script Kit from Ambion (Austin, Texas). For both probes, free isotope was removed by NucTrap Push Columns (Stratagene, La Jolla, CA). After labelling, DNAse was added to yield a concentration of 1 unit/mg DNA. The solution was incubated at 37°C for 15 min and the probe extracted with an equal volume of phenol/chloroform/isoamyl alcohol (Promega Biotec, Madison, WI). Transcription products were precipitated overnight. After centrifugation at 4°C for 15 min, the precipitate was dissolved in hydrolysis solution (40 mM sodium bicarbonate/60 mM sodium carbonate) and incubated at 60°C to reduce the probe to an average size of 150–300 bp, which was confirmed by formaldehyde–agarose gel electrophoresis.