Tissue Procurement

The periodontal tissues surrounding the teeth can be separated into the PDL, gingival tissues, and alveolar bone. Generally, the PDL tissue used in culture explants is isolated from extracted teeth. The PDL is continuous with the gingival connective tissues at the crest of the alveolar bone. Careful attention toward anatomic characteristics that discriminate the levels of root structure contained within the bone is important in separately culturing these two tissues. Otherwise, there is potential for contamination of the PDL

culture with tissues from the gingiva. By using extracted teeth to harvest PDL tissues, the bone tissue is effectively eliminated as a potential contaminating source of the tissue cultures. The extraction process usually results in the tearing of the PDL itself with a portion of the PDL remaining attached to the alveolar bone, and a portion of the PDL remaining attached to the root surface. It appears from clinical studies of both tooth avulsion / replantation, and regeneration of the attachment apparatus, that the portion of the PDL remaining on the root of an extracted tooth is critical to normal clinical healing of the attachment. Therefore, it is this tissue retained on the root surface that is cultured for in vitro investigations related to periodontal regeneration.

Once an extracted tooth has been obtained, it should be placed immediately into medium to prevent desiccation of the thin layer of tissue remaining on the root surface. Clinically, the time element with which the tissues can be maintained intact on the root surface following tooth removal, prior to successful healing with tooth replantation, has been shown to be about 30 minutes [17, 18]. Generally, we use Dulbecco's Modified Eagle's Medium (DMEM) to minimize dessication of the PDL tissue retained on the root.

The typical harvesting of PDL tissue is by mechanical scraping of the tissue remaining on the root surface following extraction. This is usually done with a sharp instrument such as a scalpel. The tissue fragments removed through this procedure are then placed into a culture system.

Before discussing culturing systems, there are several concerns that have been raised in regard to the collection of these primary tissues. These concerns include which part of the root surface is harvested, eruptive/functional status of tooth from which the tissue is collected, the age of the patient and the effect of scraping, which may remove the cementa1 layer along with the ligamentous tissue. In addition, a concern that has received considerable research effort is the difference between the connective tissues of the gingiva and the PDL.

It is apparent that in vitro cell cultures derived from these two distinct tissues, the gingiva and PDL, have significant differences [19, 20]. In order to ensure that tissue harvested from the root of an extracted tooth does not contain tissue fragments from the gingiva, the middle third of the root surface is utilized as the tissue source on periodontally healthy teeth. Likewise, the tissues in the apical third of the tooth are avoided to minimize the culturing of pulpal tissues. These apical tissues often have inflammatory changes associated with pulpal infection, quite often the cause for the extraction.

The concern over the type of tooth used as a source may be irrelevant. There is little evidence for differences in derived cultures according to the type of tooth. However, the potential for such differences has prompted some investigators to utilize only fully erupted and functionally active teeth.

The role of donor age appears to be of some significance, with older donor tissues being more fibrotic and less likely to lead to primary cultures [21]. The eruptive status has been considered to be a potentially important factor in primary culture characteristics. However, a recent study compared unerupted third molars with erupted premolars removed for orthodontic reasons and found no differences between the cultures established from these two sources [22].

The last factor to consider in establishing in vitro cell cultures using the mechanical isolation of tissue is the potential for the collection of differentiated cells lining the cementum surface of the tooth, cementoblasts [23]. These cells are capable of producing the mineralized outer root surface layer, and this process is continuous. The possibility exists that many of the tissue culture populations established from the PDL are actually derived from the cementa1 tissue. One of the more difficult aspects of explant collection is trying to separate fibroblasts derived from the PDL from cementoblasts lining the root surface. Unfortunately, there is no known marker capable of identifying these cells. At present, it is unclear which cell types are typically being cultured through this process, and one possibility is that PDL cultures demonstrating an osteoblast-like phenotype may be cultured cementoblasts.

An alternative approach to harvesting PDL has been used in a murine model. This harvesting technique is obviously impractical to use with human tissues, but is mentioned here because it presents an interesting alternative to the previous discussion. In this model, the teeth are extracted and extraction sockets allowed to heal for about one to two weeks. After this healing time, the granulation tissue within the extraction socket is removed and explanted in culture medium [24]. Earlier, it was stated that during the extraction process, the PDL tears in half and the tissue retained on the root is utilized. This model attempts to utilize the PDL tissues normally left attached to the alveolar bone. It is thought that the proliferation of cells from the remaining PDL tissue lining the extraction socket fill the extraction site during the initial healing period. It is these cells that are then harvested.

Figure 1. Phase contrast micrographs of human periodontal ligament cells showing two distinct patterns of cell growth (original magnification 10X). Top view shows whirled pattern with cell orientation in multiple directions. Bottom view shows confluent layer of cells oriented in parallel.

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