Mesenchymal Stem Cell Characteristics Of Dental Pulp And Periodontal Ligament Stem Cells After In Vivo Transplantation

In recent years, stem/progenitor cells characterized from a variety of dental relatedtissues such as gingiva, periodontal ligament (PDL), papilla, follicle and indeed, dentalpulp of both exfoliated deciduous (children’s) and adult teeth, representing a rich sourceof mesenchymal stem cells (MSCs) that are suitable for tissue engineering applicationsdue to their accessibility and multilineage differentiation capacity. In addition, dentalstem cells display multifactorial advantages such as high proliferation rate, high viabilityand easy to be induced to distinct cell lineages. Currently, dental stem cells are known tobe derived from ectomesenchymal origin and are currently considered to share a commonlineage of being derived from neural crest cells. Most, if not all, dental stem cellsidentified thus far have generic MSC-like properties, including expression of marker genes and differentiation into mesenchymal cell lineages (osteoblasts, adipocytes andchondrocytes) in vitro and, to some extent, in vivo. While extensive efforts have been andstill are being made to assess the potential of these cells in preclinical applications, twoof them, dental pulp stem cells (DPSCs) and PDL-derived stem cells (PDLSCs) havealready been tested as therapeutics in humans, although clinical trials have not beenwidely reported.The most striking feature of DPSCs and PDLSCs is their ability to regenerate dentalpulp-like and PDL-like tissues in vivo, where the local microenvironment represents animportant compartment in maintaining the stem cells status and regulates the balancebetween self-renewal and differentiation. The capacity of dental stem cells to give rise toother cell lineages, however suggests that they may have a wider clinical application. It is,however, important to consider that, although the self-renewal capability, multi-lineagedifferentiation capacity and clonogenic efficiency of DPSCs and PDLSCs have beenwell-demonstrated, their stem cell properties following in vivo transplantation remainlargely unexplored.Interestingly, there is evidence implying that DPSCs and PDLSCs maintainself-renewal capability and multipotent potential even undergoing a long-term period ofin vivo transplantation. For instance, stromal-like cells were reestablished in culture fromprimary DPSC transplants and those cells were able to generate dentin pulp-like tissueswhen retransplanted into immunocompromised mice. In a recent study, ovine PDLSCswere demonstrated to survive after8weeks post-transplantation into immunodeficientmice. These cells exhibited an immunophenotype and multipotential capacity comparableto primary PDLSCs and displayed a capacity to form fibrous ligament structures andmineralized tissues associated with vasculature in vivo, albeit at diminished levels incomparison to primary PDLSCs. Although these findings indicate the stability of dentalstem cells in an in vivo condition, at least to a certain degree, the stem cell properties ofhuman DPSCs and PDLSCs before and following in vivo implantation have yet beencontrastively evaluated. In the present study, we characterized cells obtained from invivo-regenerated dental pulp-like tissues (re-DPCs) and periodontal ligament (PDL)-like tissues (re-PDLCs) as a result of ectopic transplantation of human DPSC and PDLSCsheets, where originally obtained DPSCs and PDLSCs from dental tissues were used asmatched controls for quantitative analysis in cellular and molecular levels. Together withdata published in the literature, our data will provide additional evidence that supportsclinical utility of dental stem cells for regenerative medicine.Objective:The aim of this study was to identify the putative stem cells in regenerateddentin-pulp-like structure and periodontal ligament-like tissues.Methods:1Isolation of human dental pulp stem cells (DPSCs) and periodontal ligament stemcells (PDLSCs)Normal human third molars were collected from adults (22-28years of age) at theDental and Alveolar surgery Clinic and the use of human tissue for research wasapproved by the Institutional Review Board (IRB) of the Fourth Military MedicalUniversity School of Stomatology. Dental pulp and periodontal ligament tissues werecollected for patient-matched cell isolation and subsequent investigations. Human dentalpulp stem cells (DPSCs) and periodontal ligament stem cells (PDLSCs) were cultured aspreviously described in the literatures.2. Transplantation and Histological observation. Re-isolated cells from regenerateddental pulp-like (re-DPCs) and periodontal ligament-like tissues (re-PDLCs) andImmunofluorescence assayThe PDLSCs derived cell sheets, DPSCs derived cell pellets and human root canalfragments were prepared as previously reported. The DPSCs derived cell pellets wereinserted into the root canal space for the pulp regeneration and the PDLSCs derived cellsheets were package in the outside of the root canal fragments for the periodontal tissueregeneration respectively. After cultured in the minimal amount of cell culture mediumfor24h, the two kinds of compounds were transplanted subcutaneously into the dorsalsurface of6week-old immunodeficient mice. These procedures were approved by theAnimal Care Committee of Fourth Military Medical University and met the NIH guidelines for the care and use of laboratory animals. Each mouse received fourtransplants. The transplants were regained after60days and a fraction of them were fixedin4%paraformaldehyde overnight at4℃, decalcification with buffered10%EDTA (pH8.0) and processed for routine hematoxylin and eosin (H&E)staining for histologicalanalysis.For the culture of regenerated tissues cells, the recovered transplants were minced anddigested in a solution of3mg/mL collagenase type I and4mg/mL dispase (Sigma, StLouis, MO, USA) for1hr at37°C. The re-isolated dental pulp cells (re-DPCs) andperiodontal ligament cells (re-PDLCs) were respectively seeded in12-well plates at adensity of1×104cells/well and cultured in complete medium. When the cells achieved60%~70%confluence, removed the medium, and rinsed with PBS twice. Then the cellswere treated with4%paraformaldehyde for30minutes at room temperature and washedwith PBS again. Next, the cells were blocked non-specific interaction with10%normalgoat serum. After1h, the cells were incubated with surface of intact mitochondriaprimary antibodies (Millipore, Billerica, MA, USA) or Vimentin (GeneTex, Taiwan)primary antibodies overnight at4°C. Primary antibodies were detected withcorresponding fluorescence-conjugated secondary antibodies. Nuclei were stained withHochest (Beyotime, China). Cells were observed under an Olympus IX71fluorescencemicroscope (Olympus, Japan), and all images were captured using a TH4-200photosystem (Olympus) at200×magnification.3. The biological characteristics of the re-isolated DPSCs and PDLCsDPSCs、PDLSCs、re-DPCs and re-PDLCs were isolated and cultured by limiteddilution method and then their surface antigens were analysed by flow cytometry. Theclone formation rate and MTT assay were used to detect the cell proliferation activity ofboth cells; while the ability of cell differentiation into osteoblast and adipoblast wereevaluated by incubation of both cells in osteo-/adipo-inducing conditions; Finally, theexpression of osteogenesis related genes such as runt-related transcription factor-2(Runx2), alkaline phosphatase (ALP), bone sialoprotein(BSP), collagen type I (COL-I),runt-related transcription factor2(RUNX2) and osteocalcin (OCN)were detected by Real-Time PCR. Meanwhile, PPARγ for adipogenic differentiation and aggrecan(ACAN), collagen type II (COL-II) for chondrogenic differentiation was detectedrespectively at7days after adipogenic induced and at10days after chondrogenicinduced.4. Statistical analysis:All results are presented as the mean±standard deviation (SD) from at least threeindependent experiments. Data were analyzed by Student’s t-test or two-way analysis ofvariance (ANOVA) using SPSS software. P values less than0.05were consideredstatistically signifcant. For analysis of multiple groups, the P values were adjusted byusing the Bonferroni method.Results:It was found that the cells positive for STRO-1, CD146, CD29, CD90, and CD105wereidentifed in the regenerated dental pulp-like tissues by flow cytometry. MTT[3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assays and the clongingforming assay revealed that cultured regenerated dental pulp-like tissue cells had a strongproliferative activity and colony forming ability. Apart from this, these cells were able toform mineral deposits, lipid-containing adipocytes, and chondrocytes. However, the levelof mineralization and the role of cartilage formation in these cells were lower than that ofDPSCs. It is worth noting that the cultured regenerated periodontal tissue cells had aweakly positive for mesenchymal stem cell associated markers, a lower proliferativeactivity, and can not grow in colonies. Meanwhile, the ability of differentiation intoosteoblast, chondroblast was much weaker than PDLSCs except adipogenic capacity.Conclusions:We conclude that cells with characteristics of putative mesenchymal stem cells werefound in regenerated dental pulp-like tissues instead of in periodontal tissues, implyingDPSCs have a stronger ability of self-renewal and differentiation than PDLSCs in vivo.