The Interactions Of Patient-matched “Inflamed” And Healthy Periodontal Ligament Stem Cells In Indirect Or Direct Co-cultures

Background:Recently, with the accumulation of knowledge on periodontal ligament stem cells(PDLSCs), stem cells derived from “inflamed” periodontal ligament(PDL) tissue of periodontally diseased teeth(I-PDLSCs) have been successfully isolated and cultured as an easily accessible cell source for cell therapy with fewer ethical complications as well as increased patient acceptance than stem cells derived from healthy PDL tissue(H-PDLSCs). However, extensive evidence indicates that I-PDLSCs exhibit impaired functionalities compared to H-PDLSC in vitro and in vivo. Similarly, in our previously published findings, we isolated and characterized I-PDLSCs and H-PDLSCs from the same donor, and confirmed that I-PDLSCs indeed had an increased proliferative capacity and greater migratory potential, whereas the functionally compromised differentiation and immunomodulation and of I-PDLSCs largely limited the direct use of these cells in cytotherapy and tissue engineering. Therefore, in this study, indirect and direct co-cultureof patient-matched I-PDLSCs and H-PDLSCs were investigated to search the effective strategies to improve the therapeutic potential of I-PDLSCs in clinic. On the one hand,by observing the cell responses to conditional medias(CMs) derived from I-PDLSCs and H-PDLSCs(H-CM and I-CM), we aimed to identify that when the cell culture microenvironment of I-PDLSCs was changed as H-CM produced by patient-matched H-PDLSCs in vitro, could the osteogenic potential of I-PDLSCs be somewhat rescued?On the other hand, the patient-matched I-PDLSCs and H-PDLSCs were co-cultured in various combinations and the biological properties of these co-cultures were investigated in vitro and in vivo. We hypothesized that co-culture of I-PDLSCs directly with patient-matched H-PDLSCs may be a simple and effective strategy to obtain robust cellular materials, and I-PDLSCs could be used as adjuvant cell sources for regenerative therapies when the number of available H-PDLSCs is inadequate to generate sufficient cellular material.Methods:1. Isolation, culture and identification of patient-matched I-PDLSCs and H-PDLSCs. In order to minimize the potential interaction of allogenic cells,patient-matched I-PDLSCs and H-PDLSCs(6 samples) were isolated and identified with immunofluorescence staining.2. The interactions of patient-matched I-PDLSCs and H-PDLSCs in indirect co-cultures. “Inflamed” and healthy conditional medias(I-CM and H-CM) were prepared from the cell culture supernatant of patient-matched I-PDLSCs and H-PDLSCs respectively, and the cellular responses of these I-PDLSCs and H-PDLSCs to patient-matched I-CM and H-CM were investigated in terms of colony-forming ability,cell proliferation and adipogenic/osteogenic differentiation in vitro. There were 4 groups:H-PDLSCs incubated in H-CM(HC-HM), H-PDLSCs incubated in I-CM(HC-IM),I-PDLSCs incubated in H-CM(IC-HM) and I-PDLSCs incubated in I-CM(IC-IM),where HC-HM served as a positive control and IC-IM served as a negative control.3. The interactions of patient-matched I-PDLSCs and H-PDLSCs in direct co-cultures. Patient-matched I-PDLSCs and H-PDLSCs were co-cultured in various combinations. Cellular materials derived from these cultures were investigated regarding their proliferation, osteogenic potential, sheet formation ability and capacity to form new bone following in vivo transplantation. We established 3 co-culture test groups and 2controls, in which co-cultures consisting of 75% I-PDLSCs plus 25% H-PDLSCs, 50%I-PDLSCs plus 50% H-PDLSCs or 25% I-PDLSCs plus 75% H-PDLSCs were denoted as the 75% IC + 25% HC group, the 50% IC + 50% HC group or the 25% IC + 75% HC group, respectively. I-PDLSC monocultures(100% IC) or H-PDLSC monocultures(100% HC) served as the ‘inflamed' or healthy cell controls.4. Statistical analysis. In this study, to minimize the potential bias of manual operation, each experiment on patient-matched I-PDLSCs and H-PDLSCs was performed three times in parallel to calculate the average value. Then, the obtained data(average value) from 6 donors were further analyzed via two-way analysis of variance(ANOVA) with Tukey's post hoc test using SPSS 18.0 statistical software. The numerical results were presented as the mean ± standard deviation(SD). P<0.05 was considered statistically significant.Results:1. Both I-PDLSCs and H-PDLSCs were positive for mesenchyma stem cell(MSC)surface markers(STRO-1, CD146, CD105 and CD90), but negative for hematopoietic cell surface markers(CD31 and CD34).2. In indirect co-culture systems, I-PDLSCs and H-PDLSCs induced in either H-CM or I-CM exhibited similar adipogenic potential. However, when incubated in I-CM, both cell types demonstrated an increased capacity to proliferate but a decreased capacity to differentiate into osteoblasts. Significantly, the impaired osteogenic differentiation of I-PDLSCs was partly rescued following incubation in H-CM under osteo-inducing conditions.3. In direct co-culture systems, patient-matched I-PDLSCs and H-PDLSCs could co-exist and the proportion of I-PDLSCs tended to increase during in vitro incubation.Compared with H-PDLSC monoculture, the presence of I-PDLSCs in co-cultures appeared to enhance the overall cell proliferation. Although not totally rescued, the osteogenic and regenerative potentials of cellular materials generated by co-cultured I-PDLSCs and H-PDLSCs were significantly improved compared with those derived from I-PDLSC monocultures. Irrespective of the percentage of I-PDLSCs, robust cellular materials were obtained from co-cultures with 50% or more H-PDLSCs, which exhibited equivalent potential to form new bone in vivo compared with sheets generated by H-PDLSC monocultures.,Conclusions:1. Patient-matched I-PDLSCs and H-PDLSCs could be synchronously obtained from the same donor and both exhibited the MSC characteristics, which were the valuable research object for searching the biological properties of I-PDLSCs.2. The CMs of patient-matched I-PDLSCs and H-PDLSCs differed, and the impaired osteogenic differentiation of “inflamed” stem cells had the potential to be rescued, at least partly, for therapeutic use via changing the cell culture microenvironment in vitro.3. It was found that the co-culture of I-PDLSCs with patient-matched H-PDLSCs was a practical and effective method to obtain robust cellular materials in the following situations. In cases where H-PDLSCs are not sufficient for cell therapy, I-PDLSCs could be an alternative source, particularly when used following their co-culture with H-PDLSCs. If the obtained H-PDLSCs are adequate for use, the addition of a small portion of I-PDLSCs(e.g., lower than 25% in co-culture) may result in a large number of cells in a relatively short time.