Periodontitis is the main reason for tooth loss, and the regenerative capability of periodontal tissue is rather limited. Periodontal tissue repair is entirely dependent on implanted exogenous substitutes, though we are far from reaching the desired goal of the restoration of periodontal structure and function. The recent development of tissue engineering techniques has provided new opportunities for facilitating periodontal tissue regeneration, and the choice of seed cells is one of the key factors.
Stem cells have been a hot topic in medicine and biology in recent years and are of great importance to researchers. Adult stem cells have been found in almost all tissues, including bone marrow, cartilage, blood, nerves, muscle, fat, skin, and the cornea, intestine, liver and pancreas . PDLSCs, a type of typical adult stem cell, can be obtained from the periodontal membrane. Numerous studies have shown that PDLSCs can proliferate, differentiate, and migrate to achieve tissue regeneration when periodontal tissue is repaired under pathological conditions or external damage. Therefore, PDLSCs are considered ideal seed cells for the treatment of periodontal defects . However, to date, there are no standard criteria for the identification of PDLSCs . PDLSCs are primarily identified according to their related properties, such as cloning capacity, multi-directional differentiation in vitro, and surface molecules.
Clone formation capacity reflects important cell characteristics such as dependencies and proliferation ability. The PDLSCs in our study were able to undergo clone-like growth, and the colony-forming efficiency was approximately 15.35%, slightly lower than previously reported . We believe this difference might be associated with the cell growth status, tissue origin and culture conditions. The cytoskeleton provides a network structure in the cytoplasm that supports and maintains cell morphology and movement and is mainly composed of microfilaments, microtubules, intermediate filaments and the microtrabecular network. However, intermediate filaments differ among cells, such as keratin in epithelial cells, vimentin in mesenchymal cells and desminin muscle cells. These filaments are specific in chemical composition: they can be used as an antigen for the intermediate filament and thus be used to classify and identify cells. Immunohistochemical staining demonstrated the presence of vimentin but the absence of keratin in our PDLSCs, which indicated a mesenchymal, and not epithelial, origin for the cultured cells.
Cell growth and cell cycle status are basic parameters of cellular characteristics and are frequently used to evaluate cell viability. Previous studies have shown that the growth of PDLSCs is slower than that of other transitory proliferative cells . Nevertheless, the multiplication rate of PDLSCs improved when the tissue was repaired. The growth curve of PDLSCs in our study exhibited an S-shaped curve: the cells began to grow rapidly after 4 days of incubation and reached a plateau on the 8th day. The PDLSCs showed typical cell cycle characteristics of stem cells, with most being in the/G1 phase (75.81%) and only a few in S phase (19.80%). These results showed that the proliferation of PDLSCs was slow.
The identification of markers on the stem cell surface is important and aids in the separation, identification and analysis of stem cells. However, specific markers for PDLSCs have not been reported. Previous studies have indicated that the surface markers of PDLSCs are similar to those of bone marrow mesenchymal stem cells [27, 28]. In our study, PDLSCs were positive for expression of fibroblast surface markers (CD29, CD90, CD105) and negative for hematopoietic markers (CD34, CD45). At the same time, the PDLSCs expressed the early molecular markers of mesenchymal stem cells (CD146, STRO-1).
Stem cells have the characteristics of self-replication and multi-directional differentiation capacity. Our studies have shown that PDLSCs can be differentiated into osteoblasts and lipoblasts under the appropriate circumstances. Indeed, the multi-directional differentiation capacity of PDLSCs has great potential for clinical application in periodontal regeneration.
In the present study, we succeeded in culturing and separating original periodontal ligament stem cells using the tissue block with limiting dilution method. Furthermore, we identified stem cell properties based on cell morphology, clone formation ability, growth activity, cell surface antigens and multiple differentiation capacities.
Plaque is an important factor in the occurrence and development of periodontal disease, and P. gingivalis has been confirmed to be closely related to periodontitis . Accordingly, much research has verified that P. gingivalis can infect and invade a variety of host cell types in vitro, including primary human gingival epithelial cells , KB cells , endothelial cells , and gingival fibroblasts . P. gingivalis has been detected in gingival tissues obtained from patients with periodontitis, indicating an essential effect of invasion in the pathogenesis of periodontitis . Furthermore, the effects of P. gingivalis have also been studied in undifferentiated bone marrow stromal cells, in which P. gingivalis stimulates osteolytic cytokine expression production  via the p38 MAPK pathway , and activates a number of genes related to cell cycle arrest and apoptosis as confirmed by microarray analysis . Previous studies showed that the invasion of P. gingivalis is a rapid process, reaching completion. Imaging of infected monolayers revealed that over 90% of gingival epithelial cells were invaded by P. gingivalis after 12 min . Although the invasion efficiency did not increase after 2 h of incubation, when the interaction time was extended to 5 h, the number of internalized ecovered bacteria increased greatly because the bacteria divided within the epithelial cells . Thus, in our study, we set a 2-h time point for observing PDLSCs infection by P. gingivalis ATCC 33277.
The traditional culture method is the classical approach for detecting bacterial invasion. Invasion rates of 10% and 30% have been reported for P. gingivalis in various cell types . In our study, The PDLSC infection rates of P. gingivalis at MOIs of 50, 100, 200, and 500 were 5.83%, 8.12%, 7.77% and 7.53% according to the agar plate culture method. By q-PCR, the efficiencies of P. gingivalis infection of PDLSCs at MOIs of 50, 100, 200, and 500 were 6.74%, 10.56%, 10.36% and 9.78%, respectively. These results are similar to those of others. The infection efficiency according to the q-PCR method was higher than that according to the agar plate culture method. In addition, the difference between the agar plate culture and q-PCR methods was significant. It is not unexpected for differences between the results obtained from distinct methods. The culture assay uses intracellular survival as an alternative measure of infection and thus requires that the organisms remain viable throughout entire process. Furthermore, the culture method has a low sensitivity and is imprecise, and bacterial growth might affect the final results. The q-PCR amplification method not only has higher sensitivity than traditional culture techniques but is also less time consuming, enabling more accurate results within a shorter period of time. The optimal MOI for P. gingivalis ATCC 33277 was 100, which is similar to other results. We speculate, the bacteria have specific intake pathways at low MOIs and that at high MOIs, the cells were overwhelmed by the bacterial challenge via nonspecific mechanisms, or the interaction between P. gingivalis and PDLSCs may be related to saturation in surface receptors and signal transduction pathways.
The abilities to adhere to and invade host cells are important attributes of a successful pathogen. It has not been clearly elucidated how P. gingivalis enters in infected cells. Intracellular P. gingivalis reportedly localizes in various cellular compartments. P. gingivalis is localized in the perinuclear region of gingival epithelial cells  or in the cytoplasm in pocket epithelial cells . In contrast, P. gingivalis is not observed in the cytoplasmic spaces but replicates in endocytic vacuoles of endothelial cells . Additional studies are warranted to explain these differences, which are from the initial interactions between P. gingivalis and various types of host cells. In this study, the electron microscopic observations showed that P. gingivalis ATCC 33277 can invade PDLSCs, but the bacteria were found in the cytoplasm and were not encapsulated by endocytic vacuoles. Several bumps were patches of stretched membrane where P. gingivalis ATCC 33277 had been being endocytosed. So we suspect that the invasive properties of P. gingivalis in PDLSCs were similar to those in gingival epithelial cells. P. gingivalis exploits cellular endocytosis to enter cells, which is captured by cellular pseudopodia and invagination. Previous reports suggest that, following adhesion to the integrins, invasive event has been reported to require cytoskeleton , microtubules, integrin , lipid rafts  and bacterial fimbriae . Microorganism invasion of host cells triggers signaling pathways that rearrange the cytoskeleton, facilitating bacterial entry and aiding their survival by avoiding extracellular degradation.
Therefore, P. gingivalis is able to enter PDLSCs in this study. Additional studies are required to better understand functional changes after bacterial invasion, such as differentiation and migration of PDLSCs, the intracellular lifestyle of P. gingivalis in PDLSCs.