The Effects Of Inflammation Stimulation On Mesenchydal Stem Cells From Human Gingival Tissue And Periodontal Ligament Tissue

Background and AimPeriodontal structures contain at least4unique tissues, where two soft tissues aregingiva and periodontal ligament (PDL), while two hard tissues are alveolar bone and theroot cementum. Gingival is the oral mucosal epithelial and connective tissue that coversthe tooth around the neck and alveolar surface. PDL is a dense connective tissue thatresides between the tooth and the alveolar bone. It is well-recognized that PDL plays anintegral role at maintaining and, at a certain degree, regenerating the periodontal tissue.The identification of mesenchymal stem cells (MSCs) from both gingival tissue and PDLhas led to stem cell-based therapy a very important role to play in future periodontalregeneration. Stem cells are undifferentiated cells which exists in the organization withself-renewal and differentiation potential, and it is the basis of research tissue regeneration and reconstruction. Although PDL tissue has been demonstrated to contain multipotentstem cells, its limited source greatly limits their potential clinical application. Comparedwith the PDL tissue, the gingival tissue is easily obtained and hence, stem cells derivedfrom gingiva have attracted increasingly attention in recent years. In previous studies,MSCs have been isolated from the gingival tissue and these cells have proved to presentthe ability of self-renewal, multi-differentiation and immune regulation. However, theutility of gingival MSCs (GMSCs) as alternatives to PDL stem cells (PDLSCs), whichhave been demonstrated to be effective but with limited cell availability and reducedclinical feasibility, for periodontal regeneration in a previously diseased/inflamedenvironment remains obscure. Therefore, the aim of this study was to compare thepluripotential capacity and regenerative potential of GMSCs and PDLSCs in differentincubation conditions. We anticipate the study will provide new knowledge associatedwith the inflammation-related changes of oral MSC properties and their complications infuture periodontal tissue engineering and regeneration.MethodsFirstly, the gingival and PDL tissue were obtained from the same individuals, and thenthe primary cells were cultured. Stem cells were obtained through single cell cloningmethod and their cell surface markers were characterized by flow cytometry. Theproliferative and differentiative capacity of GMSCs and PDLSCs were contrastivelyevaluated cell growth curve, rate of colony formation, and their osteogenic, adipogenicand chondrogenic potential.Secondly, the changes of biological properties in the two types of stem cells in anartificial inflammatory microenvironment were contrastively evaluated by ALP staining,ALP quantitative, and Real-time PCR in relation to their osteogenic and adipogeniccapacity. According to the previous reports in the literature, we chose two inflammatorycytokines, i.e. IL-1β (5ng/mL) and TNF-α (10ng/mL), to build an in vitro inflammatorymicroenvironment. Finally, tranplants formed by the two stem cells (cell sheets) followingnormal or inflammatory incubation and a bone scaffold were implanted in immunodeficiency mice subcutaneous for8weeks to observe their ability of boneformation.Results1. Comparison of GMSCs and PDLSCs in terms of their isolation, proliferation anddifferentiation potential:(1) The relatively purified stem cells were obtained from the primary cells by the singlecell clonying method, and the two types of stem cells were both positive to stem cellsurface markers such as STRO-1、CD146、CD105、CD90、CD29, and negative toendothelial marker CD31and hematopoietic marker CD45. The difference of the two stemcells phenotype had no statistical significance.(2) By the comparison of the growth curve and colony-formation rate, we found thatGMSCs have stronger proliferation capacity than PDLSCs (P<0.05).(3) After osteogenic, adipogenic and chondrogenic induction in vitro, the osteogeniccapacity and chondrogenic capacities of PDLSCs were significantly higher than those ofGMSCs (P<0.05), while the adipogenic capacity of GMSCs appeared to be slightlystronger than that of PDLSCs (with no statistical difference)(P>0.05).2. Comparison of GMSCs and PDLSCs in terms of their differentiation potential innormal or inflammatory environment; the inflammatory cytokines of IL-1β (5ng/mL) andTNF-α (10ng/mL) were used to build in vitro inflammatory microenvironment and thechanges of the differentiation capacity for the two types of stem cells in response toinflammatory stimulation were evaluated.(1) The PDLSCs’ osteogenic ability was stronger than that of GMSCs in theinflammation environment (P<0.05). The osteogenic ability of the two types of stem cellswere significantly decreased in the inflammatory microenvironment, and the decrease inPDLSCs was relatively significant than that in GMSCs (P<0.01).(2) The adipogenic ability of the two types of stem cells in the inflammatoryenvironment was also suppressed, but such inhibitory effect was not statistical significant(P>0.05). The inhibition of the GMSCs was relatively stronger, and GMSCs had higher adipogenic capacity than PDLSCs in the inflammation environment (P>0.05). We canconsider that the inflammation environment have no effect on the adipogenic ability, theadipogenic ability have no difference in normal/inflammation environment.ConclusionsA high yield of MSCs with multipotent potential was successfully obtained fromhuman gingiva and PDL tissue. Both GMSCs and PDLSCs exhibited important potentialto differentiate into osteogenic, adipogenic, and chondrogenic lineages, and possessed thecapacity to generate new bone following ectopic transplantation. Human PDLSCsexhibited a more effective differentiation potential in comparison to GMSCs irrespectiveof incubation conditions, but we cannot leave aside their limited cell availability andreduced clinical feasibility. Contrastively, gingival tissue is relatively abundant and ease ofaccessibility that is generally discarded following periodontal surgery, whereby MSC-likepopulations can be obtained that are functionally equivalent to MSC-like cells from bonemarrow, PDL, or many other tissues, representing a newly identifed population ofpostnatal stem cells with immense potential in tissue regeneration. Importantly, we haveshown that an inflammation environment encompasses a decline in differentiative andregenerative potential of GMSCs and PDLSCs, but the former seemed more resistant toinflammatory incubation. This finding provides evidence for an innovative potential use ofGMSCs for periodontal regeneration in a previously diseased/inflamed periodontium.