Assay Techniques

Human PDL cells have not been well-characterized despite considerable research efforts. This is in part due to the heterogeneity between cell cultures and between sub-populations within a given cell culture, and in part to the relatively non-distinctive nature of the cells themselves. Morphologically, the cells have a typical fibroblastic appearance that with confluence can have either a parallel or whirled pattern [10] (Figure 1). It has been suggested by one study that the differences in the patterns may correlate with the phenotype of the cells. Cells cultured from the PDL have been identified as having one of two distinct phenotypes, either fibroblastic or osteoblastic [10]. The fibroblastic cells are thought to have a parallel pattern, and the osteoblastic cells are thought to have a whirled pattern.

PDL cell populations have been well characterized for collagen production, and are becoming better characterized relative to their potential for mineralizing phenotypes. PDL cell cultures have been shown to produce collagen types I, III, and V, although there was considerable variability in percentages of production between different populations [11]. There has been one report of an attempt to develop specific antibodies for PDL. Unfortunately, the specificity of the antibodies was limited, with significant cross-reactivity to other tissues [25]. Most current research is aimed at characterizing the mineralizing phenotype that is frequently found in these cell cultures. It is thought that this phenotype correlates with the cells in vivo that are responsible for the clinical outcomes associated with regeneration of the mineralized tissues of the periodontium.

This osteogenic potential has been characterized in human PDL cultures using several markers. One of the best documented characterizations has been measurement of alkaline phosphatase activity [26]. It appears that PDL cells may have increased levels of alkaline phosphatase, particularly in cultures tending toward an osteoblastic phenotype. A second measure is the cell response to 1,25-dihydroxyvitamin D3 [27, 28]. This response has been shown to include the up-regulation of osteocalcin synthesis. Osteocalcin has been shown in humans to make up a small amount (1 -2%) of the non-collagenous bone matrix, and has been identified only in bone and dentin. These findings in particular support the use of osteocalcin synthesis as an indicator of osteoblastic activity. In periodontal ligament cells, the production of osteocalcin may vary between distinct cell populations (Figure 2). In addition, the production of cAMP in response to parathyroid hormone (PTH), and the production ofmineralized matrix with long-term culture, may also be used to indicate an osteoblast-like phenotype in PDL cells [29]. In addition, there are reports documenting production of other non-collagenous bone-related proteins such as bone sialoprotein and osteonectin [27].

Figure 2. Variable expression of osteocalcin in human PDL cell cultures. Five PDL cultures were examined for osteocalcin mRNA utilizing RT-PCR techniques, with three of the cultures positive for osteocalcin (arrow indicating appropriately sized bands), and two of the cultures negative for osteocalcin. Amplified products for each of the five cultures are shown using ethidium bromide staining following gel electrophoresis (lanes 1-5).

Figure 2. Variable expression of osteocalcin in human PDL cell cultures. Five PDL cultures were examined for osteocalcin mRNA utilizing RT-PCR techniques, with three of the cultures positive for osteocalcin (arrow indicating appropriately sized bands), and two of the cultures negative for osteocalcin. Amplified products for each of the five cultures are shown using ethidium bromide staining following gel electrophoresis (lanes 1-5).

In contrast to the heterogeneity found in PDL cultures, these cultures appear to be uniform in their responsiveness to growth factors. Proliferative responses to growth factors have shown these cells to be responsive to all isoforms of PDGF, and that these cells predominantly have PDGF-a receptor subunits [12, 14]. Interestingly, there appears to be less heterogeneity between cell populations or between distinct subpopulations within a given cell population with regard to this last characteristic. The responsiveness of these cells to TGF- p has been shown to be similar to that of dermal fibroblasts, including a proliferative response at low concentrations in a delayed manner, and a down-regulation of the PDGF-a receptors at higher concentrations [30, 12].

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