Effects Of Microenvironment On Periodontal Regeneration And Differentiation Of Related Mesenchymal Stem Cells

Predicable regeneration of the attachment apparatus following periodontal disease has long been the major goal of periodontal therapy. From a biological perspective, the occurrence of this predictable periodontal regeneration depends upon two crucial factors: the availability of appropriate cell types, and a favorable local environment, that is, the presence of the cues and signals necessary to recruit and stimulate these cells. The two factors need to be precisely coordinated both temporally and spatially. The inductive microenvironment is usually composed of bioactive scaffolds or single growth factors, which are necessary for the periodontal differentiation of periodontal ligament stem cells (PDLSCs) during the periodontal tissue engineering. As we all know, the environment of periodontal regeneration is much complicated, including the participation of epithelial-mesenchymal interactions, extracellular matrix and growth factors. Therefore, it is almost impossible to achieve predictable periodontal regeneration with non-physiological inductions mediated by scaffolds or some growth factors. However, the similarity between periodontal regeneration during wound-healing and periodontal development may require that events during early cementogenesis be recapitulated. Thus based on the principles of cell and developmental biology, it can be assumed that apical tooth germ cells (APTGs) as the root/periodontal tissue (R/RT) development primordium may provide"periodontal niche"for periodontal ligament stem cells. In this paper, an indirect co-culture system of APTGs and periodontal ligament stem cells/bone marrow mesenchymal stem cells (BMMSCs) was established and apical tooth germ cell conditioned medium (APTG-CM) was used to mimic the microenvironment of R/PT development and induce the differentiateon of PDLSCs/ BMMSCs. Then on the basis of the above research, a novel"Cell Pellet"strategy was chosen for the 3-D construction of complicated structures of periodontal tissues in vivo. Our results provide an interesting progress in the periodontal engineering and present a new strategy for the ultimate clinical applications.Part.I Differentiation of periodontal regeneration-related mesenchymal stem cells induced by apical tooth germ cells.To evaluate the effects of developmental microenvironment from apical tooth germ cells on the proliferation and differentiation of PDLSCs/ BMMSCs, we established an indirect co-culture system of APTGs and PDLSCs/ BMMSCs. The data accumulated here showed that the APTG-CM, which likely contains multiple molecular signals or growth factors necessary for PDLSCs/ BMMSCs proliferation and differentiation, can effectively induce PDLSCs/ BMMSCs toward the cementoblast and osteoblast phenotype respectively and bring about the formation of cementum/PDL-like structures and bone-like structures.Taken together, it seems that the strategy that our understanding of mechanisms for R/PT (root/periodontal tissue, R/PT) development is being applied in dental tissue engineering toward predicable regeneration of periodontal tissues is practicable.Part.II Construction of a novel cell pellet and its application in periodontal tissue engineering.In this study, we proposed a novel three-dimentional cell-pellet cultivation system based on the cell sheet engineering and explored a feasible strategy to construct three-dimensional complicated structures of periodontal tissues by using cell pellets. A monolayer cell-matrix membrane (cell sheet) was constructed firstly; then the cell sheet shrank and condensed into an intact cell pellet; finally with the heterotypic layering of reshaped cell pellets, complicated periodontal structures, which include periodontal ligament, alveolar bone and cementum, were constructed. When cultured in theα-MEM medium containing 50μg/ml ascorbic acid, expanded PDLSCs/ BMMSCs synthesized a large amount of extracellular matrix and finally cell pellets with good mechanical properties were fabricated. An in vivo differentiation assay of PDLSCs/ BMMSCs pellets alone or combined with CBB/dentin slices was performed using immunodeficient mice. PDLSCs pellets alone can not produce a cementum/PDL-like structure; however, BMMSCs pellets co-cultured with APTG-CM formed bone-like tissues. The recombinations of induced PDLSCs pellets with CBB/dentin slice generated order-aligned cementum-periodontal ligament complex. Unfortunatelly, recombinations of PDLSCs/ BMMSCs pellets with dentin slices failed to contructed complicated structures of periodontal tissues.In summary, the local environment, which is composed of extracellular matrix and endogenous growth factors secrated by PDLSCs pellets, combined with the periodontal microenvironment provided by APTGs, maximally mimicked the role of HERS and basement membrane in periodontal development. Although we did not abtain the goal of fabricating 3-D complicated structures of periodontal tissues, which include periodontal ligament, alveolar bone and cementum, but this study demonstrated that our novel strategy to construct periodontal tissues is practicable and needs to be further impoved in the future.