The passage of calcitonin through the dental root : in vitro studies of diffusion kinetics and parameters affecting diffusion

Abstract

Calcitonin, a polypeptide hormone, inhibits osteoclastic activity both in vivo and in vítro. This hormone has been used in the treatment of Paget's disease, hypercalcaemia and osteoporosis. Moreover, it has been shown to both prevent experimentally-induced external inflammatory root resorption in monkey teeth (Pierce et al 1988a), and reduce pulpal inflammation (Cullum and Kline, 1985). The current study examines the notion that intracanal placement of the hormone will result in a protracted delivery to resorption sites at external root surfaces. To validate such a proposed route of therapeutic delivery, some of the features which might regulate the kinetics of calcitonin diffusion from the root canal to the periodontal membrane were examined. In an attempt to identify the contribution that mineralized matrix makes to the diffusion kinetics in dentinal tubules, the conditions have been limited to tooth material which has been freed of soft tissue by alkaline hydrolysis. Thus, to determine the status of the dentinal tubules before and after this maceration, scanning electron-microscopic (SEM) studies were undertaken. Further SEM investigations were performed to determine the effect, if any, of: i) two commonly used tooth storage media, ii) the time of storage in these media, and iii) endodontic root-canal preparation on the dentinal tubule contents in extracted teeth. These investigations were carried out in order to describe and compare the effects of these various parameters on diffusion within dentinal tubules pathways. These studies have been discussed in respect of future studies of putative therapeutic diffusants in non-macerated teeth, a situation more closely resembling that seen in vivo. Superior preservation of loose organic material in the dentinal tubules of extracted teeth was achieved: a) by storing the teeth, at 4°C, in phosphate buffered saline as compared with Dulbecco's modification of Eagle's medium, and b) by chemomechanical preparation of the root canals. The degree of preservation of loose organic material in the dentinal tubules of extracted teeth stored in either of the abovementioned media at 4°C observed after 24 h was maintained for up to 3 wk. The proportion of dentinal tubules containing loose organic contents was significantly greater in the pulpal third of the root canal under all conditions. One feature which may regulate the diffusion of calcitonin through the endodontically-prepared root canal to the external surface is the tightness of calcitonin binding to mineralized matrix. Relevant exchanges on the binding sites will provide further control over the movement of calcitonin through the dental root. This study has specifically focused on the diffusion and binding characteristics of calcitonin to the mineralized matrix of the dental root. Three experimental in vitro approaches were undertaken: (1) to establish whether calcitonin will diffuse through dentinal tubules of intact teeth from the root canal to the external environment; (2) to establish to what extent the binding coefficient of calcitonin for mineralized tooth matrix contributes to the kinetics of this process and; (3) to establish whether non-specific protein binding will exchange, inhibit or otherwise affect these diffusion kinetics. To address the first approach, intact, single-rooted premolar teeth extracted for orthodontic reasons from healthy adolescents were macerated and apically sealed. The kinetics of diffusion from the root canal to the external root surface of calcitonin was then established. Controlled standard cross-sectional diffusion exits on the tooth-roots were established by exposing windows of dentine or cementum on root surfaces which had been coated with sealant. Nail varnish, waxes, unfilled resin materials, composite resins, and poly-vinyl acetate resulted in unacceptable leakage of indicator dyes and radiolabelled calcitonin. Acrylic paint provided a satisfactory root surface seal for in excess of 2 wk. In the experimental teeth, the standard windows were either cut in the sealant so as to expose the cementum, or the cementum was cut back further to expose dentine. SEM investigation of these window preparations confirmed that the desired ultrastructural status was achieved. Teeth without windows served as controls. [I125]-calcitonin (0.04 ml) was pipetted into the root canal and the access cavity was sealed. Diffusion into standard 2 ml aliquots of PBS was monitored, as a function of time, with a garnma counter. The calcitonin appeared on the distal side of the windows by 2 h. Since material continued to appear in the PBS over the nine days of the study, it is proposed that tooth matrix might provide an intrinsic slow-release mechanism for calcitonin delivery. To establish the role of calcitonin binding in the diffusion kinetics within dentinal tubules, radioactive calcitonin was bound to ground tooth-root matrix. Both values for the saturation of binding sites and modified Scatchard plots were prepared. Macerated root matrix was also pretreated with bovine serum albumin (BSA) or nonlabelled calcitonin prior to the addition of radiolabelled calcitonin. These experiments were designed to establish what other levels of binding constraints may be relevant in this binding regulated diffusion. Briefly, binding of [I125]-labelled calcitonin to mineral could be reduced by approximately 600% by preloading the systemwith non-radiolabelled calcitonin and by about I00% where BSA was used as a competitor. The data provided us with straight lines on modified Scatchard plots, which suggest simple non-interactive binding. BSA was less competitive than non-radioactive calcitonin. It was concluded that this binding capacity contributes to diffusion kinetics which in turn will affect the levels of calcitonin detected outside the experimental window. These in vitro data suggest that an integral mechanism exists for the delivery of a slow release, localized therapeutic dose of calcitonin to external root resorption sites. Based on the data from our BSA pre-treated experiments, such a slow-release mechanism may be augmented in the presence of tissue proteins, but is nonetheless likely to be dominated by the diffusion characteristics in vivo of the dentinal tubules and cementum themselves.