The Effects Of Osthole On The Formation And Biological Properties Of Human PDLSC Or Jbmmsc Sheets

Periodontitis is a chronic infectious disease of the tooth-supporting apparatus (alveolarbone/periodontal ligament/cementum) that is a major cause for tooth loss in adults. Aprimary goal of periodontal therapy today consists not only of arresting the tissuedestruction caused by periodontal disease, but also of regenerating the tooth supportingtissues. Although conventional periodontal therapies (including scaling, root planing andperiodontal surgery among others) can arrest the disease process to a certain degree, thosetherapies have difficulties to obtain tissue regeneration.With the aim to regeneratelost/damaged periodontal tissue, a number of regenerative techniques have been developedin the past two decades, including guided tissue regeneration (GTR), bone graftingtechnology, and the use of enamel matrix derivative (EMD) and/or a wide variety ofendogenous/recombinant growth factors. However, these techniques remain insufficient capacity to stimulate complete and functional periodontal regeneration.Recently, tissue engineering represents one of the most exciting advances inregenerative medicine, and together with the recent progress in stem cell biology, it islogical to speculate that periodontal tissue may eventually be regenerated/re-established bynew advanced therapies. Since a pioneer study published in2004that demonstrated thepresence of stem cells in human periodontal ligament (PDL), generally termed PDL stemcells (PDLSCs), cell-based therapies have become a promising therapeutic choice forperiodontal regeneration. With primary success in clinically periodontal surgery thatmanages periodontal defects by administrating PDLSCs and bone marrow-derived stemcells (BMMSCs), cell-based therapy for treatment of periodontal disease has gained anincreasing interest and attention.In addition to considerations of specific cell types that may favor periodontal repair,the cell delivery method is also of critical importance in the design of cell-basedperiodontal therapy. In recent years, scaffold-free cell delivery paradigms, such as cellsheet engineering has been developed as one of the most promising research directions inthe delivery of therapeutic cells to the periodontium. Of note, Okano’s group developed atemperature-responsive culture dish that could be used to harvest cultured cellsnon-invasively as an intact sheet together with deposited extracellular matrix (ECM) thatmay preserve the cellular junctions and microenvironments in terms of various mechanical,chemical and biological properties. To date, concerted efforts have been made to make theharvest of living cell sheets more easily and more efficiency. In a latest study of our group,Vitamin C (Vc)-induced PDLSC sheet without using temperature-responsive culture dishwas found to offer an optimal therapeutic approach for periodontal tissue regeneration and,dexamethasone combined with ascorbic acid phosphate (i.e. Vc) were used to create cellsheets in vitro and enhance bone formation in vivo. Further improvements for cell sheettechnology to facilitate clinical use are required.Recently, functional ingredients from Chinese Medicine (CM) have shown to bepotential candidates to activate adult stem cells for tissue regeneration. Moreover, formore than a millennium, traditional Chinese formulations have been extensively used in clinical practice. Therefore, it is expected that those drugs can be used as useful adjuvantin cell processing technology and tissue engineering applications, sometimes as asubstitute for growth factors. In this regard, Osthole is a fruit and fundamental ingredientof Cnidium monnieri that has been demonstrated to stimulate cell proliferation andincrease cell differentiation toward osteoblast-like cells. These results suggest that Ostholemay be an effective tool for cell processing, promoting the formation of cell sheets andincreasing the overall efficiency of osteogenesis upon their use in periodontal defects.Therefore, the aim of this study was to explore the effects of Osthole on the formationcell sheets and their resultant biological properties. PDLSCs and Jaw BMMSCs(JBMMSCs) were used as the testing cells because their demonstrated capacity forperiodontal regeneration in animal models as well as in clincial cases. This study aimed toprovide the molecular basis for using small molecule compounds of Chinese Medicine incell sheet technology for future periodontal regenerative medicine.Objective:To screen the appropriate concentrations of Osthole for in vitro cultured humanPDLSCs and JBMMSCs, and to explore the biological impact of Osthole on the formationof PDLSC/JBMMSC sheets and their resultant osteogenic propertiesMethods:1. Isolation and characterization of human PDLSCs and JBMMSCs. PDLSCsand JBMMSCs were obtained by limited dilution method in vitro. The self-renewal abilityof the two types of cells was dectected by tetrazolium salt [MTT,3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide] assay and Colony-forming unit-fibroblast(CFU-F) methods, and their surface markers were analyzed by flow cytometry. Finally,their multipotent differentiation ability was identified using the adipogenic and osteogenicinduction.2. Screen the appropriate Osthole concentrations for cell culture. Four Ostholeconcentrations were designed for testing according to the literature reference:0Mol/L,10-4Mol/L,10-5Mol/L,10-6Mol/L and10-7Mol/L. Cell proliferation (detected by MTT) and cell osteogenesis ability [detected by alkaline phosphatase (ALP) staining and ALPactivity] were used as the parameters for screening the appropriate Osthole concentrationsfor cell culture.3. Effects of Osthole on the formation of PDLSC/JBMMSC sheets and theirosteogeneic potential. To determin an appropriate model of Osthole intervention for cellsheet culture (10-day culture period), four groups were designed in the present study: cellsheet culture without Osthole intervention (control group), early-stage (the first three days)intervention with Osthole (Osthole1group); Osthole intervention through the wholeculture stage (Osthole2group); last-stage (the last three days) intervention with Osthole(Osthole3group). The histologic features of cell sheets were dectected by hemotoxylinand eosin (HE) staining, scanning electron microscope (SEM),Weston-Blot, and real-timepolymerase chain reaction (RT-PCR), respectively; while ALP and alizarin red stainingand RT-PCR were used to detect the osteogenetic ability of the obtained cell sheets.4. In vivo transplantion of PDLSC/JBMMSC sheets. To evaluate the osteogenicpotential of Osthole-mediated PDLSC/JBMMSC sheets in vivo, the compounds of cellsheet/Hydroxyapatite-tricalcium phosphate（HA-TCP）were implanted subcutaneously intonude mice (cell sheets obtained without Osthole intervention were served as the controls).The transplants were harvested after four weeks post-transplantation for HE staining.5. Statistical analysis. All results are presented as the mean±standard deviation (SD)from at least three independent experiments and analyzed by a two-tailed unpaireStudent’s t test or one-way ANOVA using SPSS software. P values less than0.05wereconsidered statistically significant.Results:1. Human PDLSCs and JBMMSCs were successfully obtained in the present study;both cells showing abilities of self-proliferation and clone formation (PDLSCs16%±0.8%; JBMMSCs8.75%±0.6%). The obtained two types of cells positively expressedmesenchymal stem cell (MSC) markers such as STRO-1, CD105, CD29, CD90, andCD146, while negative for hematopoietic stem cells markers such as CD31and CD45. Following osteogenic/adipogenic induction, the calcified nodule and fat drops can be seenunder Alizarin red staining and Oil red O staining.2. MTT assay showed that a concentration of10-5Mol/L Osthole promoted theproliferation of PDLSCs and JBMMSCs more effectively in comparison to other designedconcentrations (P<0.05); ALP staining and ALP quantitative analysis also showed that10-5Mol/L was an appropriate concentration for improving cell osteogenic differentiation(P<0.05).3. The properties of PDLSC/BMMSC sheets were evaluated for determination of anappropriate model of Osthole intervention for cell sheet culture. It was found thatearly-stage (the first three days) intervention with Osthole (Osthole1group) was effectivefor JBMMSCs in terms of their relative mRNA expression, corresponding proteinproduction, and the level of ECM elements, such as Col-I, Integrin β1, and Fibronectin.However, Osthole intervention only improved Fibronectin in PDLSC sheets, irrespectiveof the models of Osthole intervention.4. The osteogenic ability of PDLSC/JBMMSC sheets was further used fordetermining the optimized model of Osthole intervention. RT-PCR results revealed thatthe mRNA levels of PDLSC sheets, such as RUNX2, OCN and ALP, were increasedsignificantly when Osthole intervention was through the whole culture stage (Osthole2group)(P<0.05). While for JBMMSC sheets, these osteogenic markers all significantlyimproved in all Osthole1groups in comparison to the control group (P<0.05).5. The results of in vivo transplantation showed that both PDLSCs/Osthole group andJBMMSCs/Osthole group formed more new bone on the surface of HA-TCP materialsthan their controls. It was also observed that the bone cells and bone lacuna on the surfaceof new bone in Osthole-intervention group. Quantitative analysis showed that theOshtole-mediated cell sheets formed more new bone than the cell sheets withoutOsthole-treated in both cell types (P<0.001); Although without Osthole intervention(control), JBMMSC sheets generated more new bone area in comparison to PDLSC sheets(P<0.05), Osthole-mediated PDLSC sheets and JBMMSC sheets showed similar newbone formation capability (P>0.05). Conclusions1. Different concentrations of Osthole had different effects on the biologicalproperties of PDLSCs and JBMMSCs. The concentration of10-5Mol/L Osthole is moreeffective for the two testing cell types in terms of cell proliferation and osteogeneticdifferentiation.2. Early-stage (the first three days) intervention with Osthole for JBMMSCs whileOsthole intervention through the whole culture stage for PDLSCs was effectively topromote the formation of PDLSC/JBMMSC sheets and their osteogenic differentiation.3. Osthole-induced PDLSC/JBMMSC sheets have an improved osteogenic abilityupon in vivo implantation.