The Effects Of Human Platelet Lysate On Mesenchymal Stem Cells Derived From Human Teeth: An Experimental Study In Vitro And In Vivo

Human platelet lysate (PL) has been known as a kind of lysate ofautologous or allogeneic platelet-rich products, and PL has been suggested as asubstitute for fetal bovine serum (FBS) in the large-scale expansion ofmesenchymal stem cells (MSCs). However, the biological effects and theoptimal concentrations of PL for the proliferation and differentiation of humanMSCs derived from teeth, such as dental pulp stem cells (DPSCs), stem cellsfrom apical papilla (SCAP), and periodontal ligament stem cells (PDLSCs)remain unexplored. We isolated, expanded and identificated DPSCs, SCAP andPDLSCs from the dental pulp, apical papilla and periodontal ligament ofextracted human teeth respectively, and evaluated the effects of PL on the cells’proliferative capacity and differentiation potential in vitro and in vivo. This willfacilitate the long-term usage of these cells for various tissue regeneration applications including pulp/dentin regeneration, bioroot formation and even forneural tissue regeneration.1. Isolation, expandation and identification of DPSCs, SCAP andPDLSCs: The isolation of human DPSCs, SCAP and PDLSCs was performedaccording to the previously reported methods, with minor modification. Whenthe cell colony formation units reached80%confluence, the cells were collectedto isolate single cell clones by limiting dilution in order to purify the cellpopulation. Immunocytochemical staining and flow cytometry-based cell sortingshowed that the cells derived from human dental pulp, apical papilla andperiodontal ligament contained mesenchymal stem cell populations. Thecapacity for differentiation into different mesenchymal tissues suggests that ourobtained cells have the key properties of MSCs.2. Platelet lysate preparation and cell culture system: On the basis of apreviously reported protocol with minor modification, human PL was preparedfrom the blood of10donors by freeze/thaw cycle. The final PL concentration(e.g.,0%,1%,5%,10%) was calculated on the basis of the volume of PL thatwas added to the total volume of the culture media. The cells were seeded onplates or HA-TCP ceramic scaffolds for the following experiments.3. The effects of PL on proliferation and differentiation of DPSCs,SCAP and PDLSCs in vitro: The proliferation of DPSCs was significantlyincreased when the cells were cultured in5%PL under all testing conditions(P<0.05). However, this enhancement was inconsistent when the cells werecultured in1%PL or in10%PL;10%PL significantly inhibited cellproliferation and was therefore excluded from further differentiation testing.Culture medium containing5%PL also significantly promoted the mineralizeddifferentiation of DPSCs, as indicated by the measurement of alkaline phosphatase activity and calcium deposition under mineral-conditioned media(P<0.05). Scanning electron microscopy and modified Ponceau trichromestaining showed that the cells treated with5%PL and mineralizing media werehighly capable of integrating with the HA/TCP biomaterials and had fullycovered the surface of the scaffold with an extensive sheet-like structure14dafter seeding. The SCAP and PDLSCs seeded on HA-TCP scaffolds inOsteogenic/odontogenic differentiation medium added with PL showedincreased proliferation, more calcium deposit and formed a more extensivesheet-like structure in the time course in vitro, and these effects were moresignificant in the PDLSCs groups.4. The effects of PL on tissue regeneration of DPSCs, SCAP andPDLSCs in vivo: The histological observations of the cell-biomaterialsconstructs or cell seeded-biomaterials in the root fragments8or12weeks afterin vivo implantation were evaluated with Van Gieson staining or modifiedPonceau trichrome staining.5%PL showed significantly positive effects ontissue regeneration of DPSCs and SCAP in two in vivo transplantation models,and the PDLSCs samples treated with5%PL/M could differentiated intocementoblast-like cells that formed a cementum-like structure, cementocyte-likecells and PDL-like tissue were also generated.In summary, our results suggest that the appropriate concentration of PLtreatment does enhance the proliferation and mineralized differentiation ofDPSCs, SCAP and PDLSCs in vitro and in vivo, which supports the use of PL asan alternative to FBS or a nonzoonotic adjuvant for cell culture in future tissueregeneration or clinical trials.