A. H. Melcher

Connective tissue cells responding to wounding of the periodontal ligament of the lower first molar in mice were studied using the techniques of radioautography and grain counting. Animals were given an intraperitoneal injection of 2 uCi 1g 3 H‐Tdr 1 hr before being killed at either 30, 72 or 120 hr after wounding. The ligament in 1 um plastic sections was divided into compartments on the basis of distance from the wound, and the relative number of labelled cells in each compartment was assessed at 30, 72 and 120 hours after wounding. The distance of each labelled cell from the closest blood vessel was also measured at each time to detect relative movement of labelled cells away from blood vessels. In a Parallel experiment, haematogenous progenitors of macrophages were depleted by irradiating the animals with 800rads prior to woundilng to determine if mistakes in identification between fibroblasts and marophages could significantly affect the results. The ultrastructural characteristics of 150 of the 3 H‐Tdr labelled cells was examined in thin sections of wounded periodontal ligament prepared for elctron microcope radioautography, The majority of cells lebelled 30 hours after wounding were confirmed to be paravascular, and most of them were found to be located within 200 μ of the wound margin. Some of these cells appreared to have divided a number of times between 30 and 72 hours after wounding, and to have migrated into the wound between 70 and 120 hours after wounding. Examination of the irradiated material and the electron microscope radio‐autographs suggested that significant numbers of macrophages had not been included in the counts of labelled cells. The elctron microscope radioautographs also suggested that cells which exhibited different degrees of cytodifferentiation had incorporated 3 H‐Tdr.

Monkey periodontal ligament fibroblasts (MPLF cells), human gingival fibroblasts (HGF cells), rat embryonic calvaria cells (REC cells), porcine periodontal ligament epithelial cells (PPLE cells) and rat osteosarcoma 17/2 cells (ROS cells) were incorporated into 3-dimensional collagen gels plated in 60 mm Petri dishes in order: first, to measure the capacity of these cell types to contract; second, to investigate cell-collagen and intercellular relationships during contraction; and third, to define the cellular contribution to tissue contraction in an in vitro system. Measurements at times up to 72 h on 3 ml gels containing 5 x 10(5) cells and with a collagen concentration of 1.20 mg/ml showed that MPLF cells contracted the gels at a significantly greater rate (P less than 0.001) than did the other cell types. In addition, contraction started sooner and was of greater extent than with the other cells. HGF cells contracted the gels more rapidly than REC and PPLE cells, while ROS cells caused no contraction. Several stages of gel compaction could be defined: (1) the attachment of cells to collagen; (2) cellular spreading within the collagen fibre matrix; (3) organization and alignment of collagen fibres by cell processes; (4) cell migration; (5) establishment of intercellular contacts; and (6) the development of a cellular reticular arrangement within the gel and the extension of this arrangement into a 3-dimensional, tissue-like, honeycomb network. Electron microscopic observations on 0.1 ml gels containing MPLF cells showed that, in the early contractile phase, numerous cell processes attached to or enclosed collagen fibrils. These processes contained microfilamentous material and few organelles. In compacted gels, the cells contained an increased amount of distended rough endoplasmic reticulum and Golgi membranes. Since MPLF cells have the capacity for vigorous contraction of the collagen gels and since they develop a reticular, 3-dimensional structure in compacted gels that is reminiscent of the relationship of periodontal ligament fibroblasts to collagen fibres in vivo, it is suggested that they could provide the major force necessary for tooth eruption in vivo. This system also provides a well-defined in vitro model to study the sequential stages that occur during contraction processes.