Mesenchymal stem cells from Wharton's Jelly and periodontal ligament: reliable not controversial sources for osteogenic differentiation and regenerative medicine.

Mesenchymal stem cells (MSCs) are uniquely capable of crossing germinative layers borders (these cell populations are able to differentiate towards ectoderm-, mesoderm- and endoderm- derived lineages) and are viewed as promising cells for regenerative medicine approaches in several diseases. Some undoubtedly limiting factors for the clinical use of MSCs, i.e. for the repair of bone defects, are related to many different problems, that only partially today can be overcome through ex-vivo expansion and cells modification strategies. Considering MSCs sources, the use of fetal annexes-derived MSCs, such as MSCs from Whartonʼs jelly of umbilical cord (WJMSCs), or the recruitment of MSCs from “stem cell niches” in adult discard tissues, like periodontal ligament (PDL) of extracted teeth (PDLMSCs), could be promising in compensating limits of MSCs traditional sources, i.e. Bone Marrow, like small cells number, high harvesting technique morbility, difficult cells commitment due to early senescence. Uniforming and investigating best cells culture conditions with in vitro different experimental strategies is useful in our job to understand and control differentiation mechanisms and finally to influence the yield and proliferation rate of these MSCs populations, together with their osteogenic potential. The aim of our study is briefly sumarized: - To isolate and culture MSCs cells from human Umbilical Cord Whartonʼs Jelly and Periodontal Ligament; - To compare characteristics between all samples recruited and to link them to clinical aspects of tissue donors; - To characterise both MSCs population in regard to their proliferation and differentiation potential; - To investigate their functional characteristics before and after alginate microbeads encapsulation; - To investigate effects of three-dimensional systems and microgravity on MSCs before and after differentiation. WJMSCs and PDLSCs were analyzed for the expression of MSC markers, and then committed to osteogenic differentiation. Before and after differentiation, alkaline phosphatase (ALP) activity, the expression level of a specific osteoblast transcription factor (Runx2), and mineralization status were evaluated. The performance of WJMSCs and PDLSCs was then compared in 3-D culture systems (alginate beads and bioreactor system) in terms of viability, proliferation, secretive profile, expression of markers and effectiveness of phenotype modulation. Characterization of MSCs population was succesfull for all samples investigated, and for largest samples cohort (WJMSCs) it was possible to correlate cells biochemical parameters to clinical donors features. These findings may help during selection of best donors to combine them with scaffolds and biomaterials to promote tissue regeneration. For both MSCs populations, we demonstrated that cells can live and grow in 3- D systems. All MSCs samples analyzed showed a substantial osteogenic potential, before and after encapsulation in alginate microbeads. Modulation of cells culture conditions, with the use of nanotechnologies strategies or the use of bioreactors, like Rotary Cell Culture SystemTM (RCCS-4TM bioreactor, SyntheconTM, Inc., Houston, TX, U.S.A.) with High Aspect Ratio Vessel (HARVTM) has been shown to be a useful and promising approach to investigate characteristics and environmental effects of cells/ biomaterials combinations, in order to predict their effect and potential for regenerative medicine strategies.