| Background:Recently, stem cells derived from “inflamed” tissue of teeth with periodontitis(I-PDLSCs) might represent an easily accessible source of mesenchyma stem cells(MSCs) with fewer ethical disputes and increased acceptance by patients as cell therapy and was considered as a promising source of MSCs for tissue engineering and regenerative medicine, compared to stem cells derived from healthy periodontal ligament(PDL) tissue(H-PDLSCs). With the accumulation of knowledge on I-PDLSCs, certain investigators have suggested that the stem cell properties of I-PDLSCs are similar to those of H-PDLSCs, whereas others have reported that the I-PDLSCs are highly dysfunctional in terms of their osteogenic potential and immunomodulatory properties due to in vivo inflammatory environment. Alone or in combination, differences in the sample selection(e.g., age, gender, sample size,inclusion/exclusion criteria and sampling method) may contribute to such contradictions. Hence, to minimize these potential interferences, this study has tried to isolate and culture I-PDLSCs and H-PDLSCs from the same donor and to further confirm the cellular characteristics of I-PDLSCs via a patient-matched comparison in vitro and in vivo, in terms of cell proliferation,differentiation and immunomodulatory analyses, providing reliable research basis for the future study on I-PDLSCs and their potential application in tissue regeneration.Methods:1. Isolation, culture and identification of patient-matched I-PDLSCs and H-PDLSCs. Firstly, Patients with at least one tooth that was irreversibly affected by incurable periodontitis and at least one periodontally healthy third molar extracted due to impaction or for reasons related to tooth dysfunction were selected. “Inflamed” and healthy PDL were gently scraped from the surface of patient-matched tooth with periodontitis and healthy tooth, and then were digested with protease. Finally, The obtained I-PDLSCs and H-PDLSCs(n=6) were performed in terms of stem cell markers,cell proliferation, cell migration and multiple differentiation potential.2. Immunomodulatory analyses of patient-matched I-PDLSCs and H-PDLSCs.Peripheral blood mononuclear cells(PBMNCs) were extracted from the fresh blood obtained from three systemically healthy volunteers respectively, and I-PDLSCs or H-PDLSCs were co-cultured with PBMNCs at different proportions for certain time to investigate the effects of PDLSCs on the proliferation, apoptosis and cytokine production of PBMNCs in vitro.3. Evaluation of I-PDLSC and H-PDLSC sheet functionalities. After 10 days of cell-sheet induction to form I-PDLSC sheets or H-PDLSC sheets, one part of cell sheets were subjected to quality analysis in vitro, including morphological detection and multiple differentiation potential assays; another part of cell sheets were transplanted into the subcutaneous site in the dorsal region of immunodeficient mice,after 8 weeks, the transplants were harvested to analyze the in vivo bone regenerative capacity of PDLSC sheets.4. Statistical analyses. In order to minimize the potential bias of manual operation, all experiments on cells derived from each donor(the patient-matched I-PDLSCs and H-PDLSCs) were performed at least three repeated times and recorded as the average for the following statistical analysis. Then, the obtained data(n=6) were further analyzed using a paired-sample t-test via SPSS software 18.0. All numerical values are presented as the mean ± standard deviation(SD) and P < 0.05 indicates a significant difference between the patient-matched I-PDLSCs and H-PDLSCs.Results:1. Patient-matched I-PDLSCs and H-PDLSCs were successfully obtained from each donor. Both cells were positive for vimentin(the specific intermediate-filament protein of mesenchymal cells) and MSC markers(STRO-1, CD146, CD105 and CD44), but negative for cytokeratin(the specific intermediate-filament protein of epithelial cells) and hematopoietic cell markers(CD45 and CD34). In addition, although both I-PDLSCs and H-PDLSCs demonstrated similar adipogenic potential, I-PDLSCs exhibited an increased capacity to proliferate, a greater potential to migrate and a decreased capacity to differentiate into osteoblasts in vitro.2. When I-PDLSCs and H-PDLSCs were co-cultured with PBMNCs at different proportions for certain time, both cell types exhibited immunosuppression of PBMNC proliferation and I-PDLSCs appeared to be significantly less effect than H-PDLSCs until the cells were co-cultured for longer than 72 h(P < 0.05 or 0.01). The percentages of PBMNC apoptosis following 72 h of co-culture with I-PDLSCs appeared to be less than that of H-PDLSCs. At the same time, although both cell types suppressed the generation of cytokines by PBMNCs, I-PDLSCs showed impaired immunomodulation in terms of their suppression on interleukin(IL)-2, tumor necrosis factor(TNF)-? and interferon (IFN)-? production(P < 0.05 or 0.01). Interestingly, with regard to IL-10 production,there was no significant difference between I-PDLSCs and H-PDLSCs.3. After 10 days of cell-sheet induction, both I-PDLSCs and H-PDLSCs formed typical membrane-like cell sheets. The thickness and living cells of I-PDLSC sheets were substantially lower than that of H-PDLSC sheets(P < 0.05 or 0.01), but compared with H-PDLSC sheets, I-PDLSCs appeared to produce higher levels of collagen(COL)-1,periostin and integrin ?1 than did H-PDLSCs(relative to the total protein content).Similar to the data obtained at the cellular level, I-PDLSC sheets exhibited a significantly lower osteogenic and chondrogenic potential than H-PDLSC sheets. Moreover, the percentage area of new bone tissue generated by I-PDLSC sheets appeared to be significantly less than that of H-PDLSC sheets in vivo(P < 0.05).Conclusions:1. The obtained data in the present study provide additional evidence that although I-PDLSCs are functionally compromised compared with H-PDLSCs, they retain MSC characteristics and have the potential to form typical mineralized nodule and bone tissue in vitro and in vivo. Hence, I-PDLSCs may provide an alternative source of MSCs for periodontal tissue regeneration. In addition, the changes on biological properties of I-PDLSCs(cell proliferation and migration) by in vivo inflammatory environment warrant further exploration.2. Comparative analysis on patient-matched I-PDLSCs and H-PDLSCs provides a valuable research method to further study the essential feature of I-PDLSCs in the future... |
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