Enhanced VEGF/VEGF-R and RUNX2 Expression in Human Periodontal Ligament Stem Cells Cultured on Sandblasted/Etched Titanium Disk.

Bone formation, in skeletal development or in osseointegration processes, is the result of interaction between angiogenesis and osteogenesis. To establish osseointegration, cells must attach to the implant in a direct way without any deposition of soft tissue. Structural design and surface topography of dental implants enhance the cell attachment and can affect the biological response. The aim of the study was to evaluate the cytocompatibility, osteogenic and angiogenic markers involved in bone differentiation of human periodontal ligament stem cells (hPDLSCs) on different titanium disks surfaces. The hPDLSCs were cultured on pure titanium surfaces modified with two different procedures, sandblasted (Control-CTRL) and sandblasted/etched (Test-TEST) as experimental titanium surfaces. After 1 and 8 weeks of culture VEGF, VEGF-R, and RUNX2 expression was evaluated under confocal laser scanning microscopy. To confirm the obtained data, RT-PCR and WB analyses were performed in order to evaluate the best implant surface performance. TEST surfaces compared to CTRL titanium surfaces enhanced cell adhesion and increased VEGF and RUNX2 expression. Moreover, titanium TEST surfaces showed a different topographic morphology that promoted cell adhesion, proliferation, and osteogenic/angiogenic commitment. To conclude, TEST surfaces performed more efficiently than CTRL surfaces; furthermore, TEST surface results showed them to be more biocompatible, better tolerated, and appropriate for allowing hPDLSC growth and proliferation. This fact could also lead to more rapid bone-titanium integration.

Functional Relationship between Osteogenesis and Angiogenesis in Tissue Regeneration.

Bone tissue renewal can be outlined as a complicated mechanism centered on the interaction between osteogenic and angiogenic events capable of leading to bone formation and tissue renovation. The achievement or debacle of bone regeneration is focused on the primary role of vascularization occurrence; in particular, the turning point is the opportunity to vascularize the bulk scaffolds, in order to deliver enough nutrients, growth factors, minerals and oxygen for tissue restoration. The optimal scaffolds should ensure the development of vascular networks to warrant a positive suitable microenvironment for tissue engineering and renewal. Vascular Endothelial Growth Factor (VEGF), a main player in angiogenesis, is capable of provoking the migration and proliferation of endothelial cells and indirectly stimulating osteogenesis, through the regulation of the osteogenic growth factors released and through paracrine signaling. For this reason, we concentrated our attention on two principal groups involved in the renewal of bone tissue defects: the cells and the scaffold that should guarantee an effective vascularization process. The application of Mesenchymal Stem Cells (MSCs), an excellent cell source for tissue restoration, evidences a crucial role in tissue engineering and bone development strategies. This review aims to provide an overview of the intimate connection between blood vessels and bone formation that appear during bone regeneration when MSCs, their secretome-Extracellular Vesicles (EVs) and microRNAs (miRNAs) -and bone substitutes are used in combination.

3D Printing PLA/Gingival Stem Cells/ EVs Upregulate miR-2861 and -210 during Osteoangiogenesis Commitment.

Bone tissue regeneration strategies require approaches that provide an osteogenic and angiogenic microenvironment able to drive the bone growth. Recently, the development of 3D printing biomaterials, including poly(lactide) (3D-PLA), enriched with mesenchymal stem cells (MSCs) and/or their derivatives, such as extracellular vesicles (EVs) has been achieving promising results. In this study, in vitro results showed an increased expression of osteogenic and angiogenic markers, as RUNX2, VEGFA, OPN and COL1A1 in the living construct 3D-PLA/human Gingival MSCs (hGMSCs)/EVs. Considering that EVs carry and transfer proteins, mRNA and microRNA into target cells, we evaluated miR-2861 and miR-210 expression related to osteoangiogenesis commitment. Histological examination of rats implanted with 3D-PLA/hGMSCs/EVs evidenced the activation of bone regeneration and of the vascularization process, confirmed also by MicroCT. In synthesis, an upregulation of miR-2861 and -210 other than RUNX2, VEGFA, OPN and COL1A1 was evident in cells cultured in the presence of the biomaterial and EVs. Then, these results evidenced that EVs may enhance bone regeneration in calvaria defects, in association with an enhanced vascularization offering a novel regulatory system in the osteoangiogenesis evolution. The application of new strategies to improve biomaterial engraftment is of great interest in the regenerative medicine and can represent a way to promote bone regeneration.

MRAP2 regulates endometrial receptivity and function.

A successful embryo implantation depends on the synchronization between a competent blastocyst and a receptive endometrium. Recently, potential modulators of endometrial receptivity (OVGP1, MRAP2, ZCCHC12, and HAP1) have been reported likely with a functional role during embryo implantation. The aim of this study was to evaluate the gene expression of these genes in the endometrium of infertile women. Eighteen endometrial biopsies, during secretory lutheal phase, were recruited from women with unexplained infertility and women who cannot conceive due to their partners' fertility problems. qRT-PCR was carried out to evaluate MRAP2, OVGP1, ZCCHC12 and HAP1 gene expression. MRAP2 expression was also detected by western blot and it was localized by immunohistochemistry. Morphological analysis was performed by light microscopy. MRAP2 was significantly up-regulated in study vs. control group. Western blot analysis confirmed the observed MRAP2 up-expression. MRAP2 resulted mainly localized in the epithelial cells of uterine glands. Morphological analysis displayed that the epithelium of the uterine glands undergo hypertrophy in women with unexplained infertility in respect to women with male infertility factor. MRAP2 could be considered a mediator of endometrial receptivity likely acting on endometrial stability by binding to MCRs and PKR1.

5-Aza Exposure Improves Reprogramming Process Through Embryoid Body Formation in Human Gingival Stem Cells.

Embryoid bodies (EBs) are three-dimensional aggregates formed by pluripotent stem cells, including embryonic stem cells and induced pluripotent stem cells. They are used as an in vitro model to evaluate early extraembryonic tissue formation and differentiation process. In the adult organisms, cell differentiation is controlled and realized through the epigenetic regulation of gene expression, which consists of various mechanisms including DNA methylation. One demethylating agent is represented by 5-Azacytidine (5-Aza), considered able to induce epigenetic changes through gene derepression. Human gingival mesenchymal stem cells (hGMSCs), an easily accessible stem cells population, migrated from neural crest. They are particularly apt as an in vitro study model in regenerative medicine and in systemic diseases. The ability of 5-Aza treatment to induce hGMSCs toward a dedifferentiation stage and in particular versus EBs formation was investigated. For this purpose hGMSCs were treated for 48 h with 5-Aza (5 µM). After treatment, hGMSCs are organized as round 3D structures (EBs-hGMSCs). At light and transmission electron microscopy, the cells at the periphery of EBs-hGMSCs appear elongated, while ribbon-shaped cells and smaller cells with irregular shape surrounded by extracellular matrix were present in the center. By RT-PCR, EBs-hGMSCs expressed specific transcription markers related to the three germ layers as MAP-2, PAX-6 (ectoderm), MSX-1, Flk-1 (mesoderm), GATA-4, and GATA-6 (endoderm). Moreover, in EB-hGMSCs the overexpression of DNMT1 and ACH3 other than the down regulation of p21 was detectable. Immunofluorescence staining also showed a positivity for specific etodermal and mesodermal markers. In conclusion, 5-Aza was able to induce the direct conversion of adult hGMSCs into cells of three embryonic lineages: endoderm, ectoderm, and mesoderm, suggesting their possible application in autologous cell therapy for clinical organ repair.

A novel role in skeletal segment regeneration of extracellular vesicles released from periodontal-ligament stem cells.

PURPOSE: The combination of oral derived stem cells and 3-D scaffolds is considered advantageous in bone repair. In particular, collagen membranes possess ideal biological properties and can support infiltration and proliferation of osteoblasts, promoting bone regeneration. Our study aimed to develop a new biocompatible osteogenic construct composed of a commercially available collagen membrane (Evolution [Evo]), human periodontal-ligament stem cells (hPDLSCs) enriched with extracellular vesicles (EVs), or polyethylenimine (PEI)-engineered EVs (PEI-EVs). METHODS: Osteogenic ability and expression of osteogenic genes were evaluated in vitro in hPDLSCs cultured with or without Evo, with Evo and EVs, or PEI-EVs. In addition, the bone-regeneration capacity of Evo, Evo enriched with hPDLSCs, Evo enriched with hPDLSCs and EVs/PEI-EVs was investigated in rats subjected to calvarial defects. RESULTS: Our results showed that Evo enriched with EVs and PEI-EVs showed high biocompatibility and osteogenic properties in vitro and in vivo. In addition, quantitative reverse-transcription polymerase chain reaction demonstrated the upregulation of osteogenic genes, such as TGFB1, MMP8, TUFT1, TFIP11, BMP2, and BMP4, in the presence of PEI-EVs. Upregulation of BMP2/4 was confirmed for Evo enriched with PEI-EVs and hPDLSCs both in vitro by Western blot and in vivo by immunofluorescence. CONCLUSION: Our results indicated that Evo enriched with hPDLSCs and PEI-EVs is able to promote a bone-regeneration process for the treatment of calvarium and ossification defects caused by accidental or surgery trauma. In particular, PEI-EVs had a significant role in activation of the osteogenic process.

Three-dimensional printed PLA scaffold and human gingival stem cell-derived extracellular vesicles: a new tool for bone defect repair.

BACKGROUND: The role of bone tissue engineering in the field of regenerative medicine has been a main research topic over the past few years. There has been much interest in the use of three-dimensional (3D) engineered scaffolds (PLA) complexed with human gingival mesenchymal stem cells (hGMSCs) as a new therapeutic strategy to improve bone tissue regeneration. These devices can mimic a more favorable endogenous microenvironment for cells in vivo by providing 3D substrates which are able to support cell survival, proliferation and differentiation. The present study evaluated the in vitro and in vivo capability of bone defect regeneration of 3D PLA, hGMSCs, extracellular vesicles (EVs), or polyethyleneimine (PEI)-engineered EVs (PEI-EVs) in the following experimental groups: 3D-PLA, 3D-PLA + hGMSCs, 3D-PLA + EVs, 3D-PLA + EVs + hGMSCs, 3D-PLA + PEI-EVs, 3D-PLA + PEI-EVs + hGMSCs. METHODS: The structural parameters of the scaffold were evaluated using both scanning electron microscopy and nondestructive microcomputed tomography. Nanotopographic surface features were investigated by means of atomic force microscopy. Scaffolds showed a statistically significant mass loss along the 112-day evaluation. RESULTS: Our in vitro results revealed that both 3D-PLA + EVs + hGMSCs and 3D-PLA + PEI-EVs + hGMSCs showed no cytotoxicity. However, 3D-PLA + PEI-EVs + hGMSCs exhibited greater osteogenic inductivity as revealed by morphological evaluation and transcriptomic analysis performed by next-generation sequencing (NGS). In addition, in vivo results showed that 3D-PLA + PEI-EVs + hGMSCs and 3D-PLA + PEI-EVs scaffolds implanted in rats subjected to cortical calvaria bone tissue damage were able to improve bone healing by showing better osteogenic properties. These results were supported also by computed tomography evaluation that revealed the repair of bone calvaria damage. CONCLUSION: The re-establishing of the integrity of the bone lesions could be a promising strategy in the treatment of accidental or surgery trauma, especially for cranial bones.

Biofunctionalized Scaffold in Bone Tissue Repair.

Bone tissue engineering is based on bone grafting to repair bone defects. Bone graft substitutes can contribute to the addition of mesenchymal stem cells (MSCs) in order to enhance the rate and the quality of defect regeneration. The stem cell secretome contains many growth factors and chemokines, which could affect cellular characteristics and behavior. Conditioned medium (CM) could be used in tissue regeneration avoiding several problems linked to the direct use of MSCs. In this study, we investigated the effect of human periodontal ligament stem cells (hPDLSCs) and their CM on bone regeneration using a commercially available membrane scaffold Evolution (EVO) implanted in rat calvarias. EVO alone or EVO + hPDLSCs with or without CM were implanted in Wistar male rats subjected to calvarial defects. The in vivo results revealed that EVO membrane enriched with hPDLSCs and CM showed a better osteogenic ability to repair the calvarial defect. These results were confirmed by acquired micro-computed tomography (CT) images and the increased osteopontin levels. Moreover, RT-PCR in vitro revealed the upregulation of three genes (Collagen (COL)5A1, COL16A1 and transforming growth factor (TGF)ß1) and the down regulation of 26 genes involved in bone regeneration. These results suggest a promising potential application of CM from hPDLSCs and scaffolds for bone defect restoration and in particular for calvarial repair in case of trauma.

Endothelial committed oral stem cells as modelling in the relationship between periodontal and cardiovascular disease.

In the present study we have mimicked, in vitro, an inflammatory process using Lipopolysaccharide derived from Porphyromonas Gingivalis (LPS-G) and human Periodontal Ligament Stem Cells induced to endothelial differentiation (e-hPDLSCs). The research project has been organized into the three following steps: i) induction of hPDLSCs toward endothelial differentiation; ii) evaluation of the molecular signaling pathway involved in the response to the LPS-G, and iii) functional response evaluation of the living construct constituted by porcine decellularized valve/e-hPDLSCs treated with LPS-G. Obtained results showed that 5 µg/ml LPS-G stimulus provokes: a slowdown of cell growth starting from 24 hr and the release of IL6, IL8, and MCP1 molecules. Signaling network analyzed showed the activation of TLR4/ NFkB/ERK1/2/p-ERK1/2 signaling mediated by MyD88 in LPS-G stimulated e-hPDLSCs, moreover a time course put in evidence a nuclear traslocation of ERK1/2 and p-ERK1/2 in differentiated samples. Following, the ability of e-hPDLSCs to expand and colonize the decellularized porcine heart valves was appraised at ultrastructural level. Considering that, the Reactive Oxygen Species (ROS) play an important role in the progression and development of cardiovascular disease (CVD), in LPS-G living construct model e-hPDLSCs/decellularized porcine heart valves (dPHV), ROS production was assessed. Time lapse experiments evidenced that LPS-G provokes in e-hPDLSCs a rapid and sustained increase in ROS generation, negligible on undifferentiated cells. From obtained data, by multiparametric analyses, a reasonable conclusion may be that the inflammation process activated by LPS-G can affect endothelial cells and could represent in vivo a possible pathological and predictor state of CVD.

Biotherapeutic Effect of Gingival Stem Cells Conditioned Medium in Bone Tissue Restoration.

Bone tissue engineering is one of the main branches of regenerative medicine. In this field, the use of a scaffold, which supported bone development, in combination with mesenchymal stem cells (MSCs), has promised better outcomes for bone regeneration. In particular, human gingival mesenchymal stem cells (hGMSCs) may present advantages compared to other MSCs, including the easier isolation. However, MSCs' secretome has attracted much attention for its potential use in tissue regeneration, such as conditioned medium (CM) that contains different soluble factors proved to be useful for the regenerative purposes. In this study, we evaluated the osteogenic capacity of a poly-(lactide) (3D-PLA) scaffold enriched with hGMSCs and hGMSCs derived CM and its ability to regenerate bone defects in rat calvarias. 3D-PLA alone, 3D-PLA + CM or 3D-PLA + hGMSCs with/without CM were implanted in Wistar male rats subjected to calvarial defects. We observed that 3D-PLA scaffold enriched with hGMSCs and CM showed a better osteogenic capacity, being able to repair the calvarial defect as revealed in vivo by morphological evaluation. Moreover, transcriptomic analysis in vitro revealed the upregulation of genes involved in ossification and regulation of ossification in the 3D-PLA + CM + hGMSCs group. All of these results indicate the great osteogenic ability of 3D-PLA + CM + hGMSCs supporting its use in bone regenerative medicine, in particular in the repair of cranial bone defects. Especially, hGMSCs derived CM played a key role in the induction of the osteogenic process and in bone regeneration.

Porphyromonas gingivalis lipopolysaccharide stimulation in human periodontal ligament stem cells: role of epigenetic modifications to the inflammation.

Periodontitis is a chronic oral inflammatory disease produced by bacteria. Gingival retraction and bone and connective tissues resorption are the hallmarks of this disease. Chronic periodontitis may contribute to the risk of onset or progression of neuroinflammatory pathological conditions, such as Alzheimer's disease. The main goal of the present study was to investigate if the role of epigenetic modulations is involved in periodontitis using human periodontal ligament stem cells (hPDLSCs) as an in vitro model system. hPDLSCs were treated with lipopolysaccharide of Porphyromonas gingivalis and the expression of proteins associated with DNA methylation and histone acetylation, such as DNMT1 and p300, respectively, and inflammatory transcription factor NF-kB, were examined. Immunofluorescence, Western blot and next generation sequencing results demonstrated that P. gingivalis lipopolysaccharide significantly reduced DNA methylase DNMT1, while it markedly upregulated the level of histone acetyltransferase p300 and NF-kB in hPDLSCs. Our results showed that P. gingivalis lipopolysaccharide markedly regulate the genes involved in epigenetic mechanism, which may result in inflammation induction. We propose that P. gingivalis lipopolysaccharide-treated hPDLSCs could be a potential in vitro model system to study epigenetics modulations associated with periodontitis, which might be helpful to identify novel biomarkers linked to this oral inflammatory disease.

MyD88/ERK/NFkB pathways and pro-inflammatory cytokines release in periodontal ligament stem cells stimulated by Porphyromonas gingivalis.

The present study was aimed at investigating whether human Periodontal Ligament Stem Cells (hPDLSCs) were capable of sensing and reacting to lipopolysaccharide from Porphyromonas gingivalis (LPS-G) which is widely recognized as a major pathogen in the development and progression of periodontitis. At this purpose hPDLCs were stimulated with 5 µg/mL LPS-G various times and the expression of toll-like receptor 4 (TLR4) was evaluated. Toll-like receptors (TLRs) play an essential role in innate immune signaling in response to microbial infections, and in particular TLR4, type-I transmembrane proteins, has been shown recognizing LPS-G. Our results put in evidence, in treated samples, an overexpression of TLR4 indicating that, hPDLSCs express a functional TLR4 receptor. In addition, LPS-G challenge induces a significant cell growth decrease starting from 24 h until 72 h of treatment. LPS-G leads the activation of the TLR4/MyD88 complex, triggering the secretion of proinflammatory cytokines cascade as: IL-1α, IL-8, TNF-α and ß and EOTAXIN. Moreover, the upregulation of pERK/ERK signaling pathways and NFkB nuclear translocation was evident. On the basis of these observations, we conclude that hPDLSCs could represent an appropriate stem cells niche modeling leading to understand and evaluate the biological mechanisms of periodontal stem cells in response to LPS-G, mimicking in vitro an inflammatory process occurring in vivo in periodontal disease.

Stemness Maintenance Properties in Human Oral Stem Cells after Long-Term Passage.

Background. Neural crest-derived mesenchymal stem cells (MSCs) from human oral tissues possess immunomodulatory and regenerative properties and are emerging as a potential therapeutic tool to treat diverse diseases, such as multiple sclerosis, myocardial infarction, and connective tissue damages. In addition to cell-surface antigens, dental MSCs express embryonic stem cell markers as neural crest cells originate from the ectoderm layer. In vitro passages may eventually modify these embryonic marker expressions and other stemness properties, including proliferation. In the present study, we have investigated the expression of proteins involved in cell proliferation/senescence and embryonic stem cell markers during early (passage 2) and late passages (passage 15) in MSCs obtained from human gingiva, periodontal, and dental pulp tissues. Methods. Cell proliferation assay, beta galactosidase staining, immunocytochemistry, and real-time PCR techniques were applied. Results. Cell proliferation assay showed no difference between early and late passages while senescence markers p16 and p21 were considerably increased in late passage. Embryonic stem cell markers including SKIL, MEIS1, and JARID2 were differentially modulated between P2 and P15 cells. Discussion. Our results suggest that the presence of embryonic and proliferation markers even in late passage may potentially endorse the application of dental-derived MSCs in stem cell therapy-based clinical trials.

Conditioned medium from relapsing-remitting multiple sclerosis patients reduces the expression and release of inflammatory cytokines induced by LPS-gingivalis in THP-1 and MO3.13 cell lines.

The present research was aimed at evaluating the effect of the conditioned medium (CM) from human periodontal ligament stem cells (hPDLSCs) obtained from healthy donors (hPDLSCs-CM) and from Relapsing-Remitting Multiple Sclerosis patients (RR-MS-CM) on inflammatory response induced by Porphyromonas gingivalis lipopolysaccharide (LPS-G) in a monocytoid human cell line (THP-1) and human oligodendrocyte cell line (MO3.13). Human periodontal ligament biopsies were carried out from control donor patients and selected RR-MS donors. Sample tissues were obtained from premolar teeth during root scaling and subsequently cultured. The effect of hPDLSCs-CM and RR-MS-CM on cell viability in PMA differentiated THP-1 (as a model of microglia) was measured using a 3-[4,5-dimethylthiazol-2-yl]-2,5 diphenyltetrazolium bromide (MTT) assay. The same experiments were performed in undifferentiated and differentiated MO3.13 cells used as models of progenitor cells and oligodendrocytes, respectively. The expression of tumor necrosis factor alpha (TNF)-α, interleukin (IL)-1ß and IL-6 was evaluated by Real-Time Polymerase Chain Reaction (RT-PCR), and enzyme-linked immunosorbent assay (ELISA). The expression level of the Toll-like receptor 4 (TLR-4), for which LPS-G is a ligand, was evaluated by Western blot analysis. The results were analyzed by ANOVA using Graph Pad Prism software. LPS-G significantly increased TNFα, IL-1ß and IL-6 mRNA expression and protein levels in the differentiated THP-1 cells and oligodendrocyte MO3.13 progenitor cells. Treatment with hPDLSCs-CM or with RR-MS-CM significantly attenuated the LPS-induced expression and production of these pro-inflammatory cytokines. The CM from both healthy donors and RR-MS patients also reduced the LPS-G stimulated protein levels of TLR-4 in differentiated THP-1 cells. On the whole our data add new evidence on the anti-inflammatory effects of these peculiar stem cells even when derived from RR-MS patients and open novel perspectives in the therapeutic use of autologous periodontal stem cells in neuroinflammatory/neurodegenerative diseases including MS.

Stemness Characteristics of Periodontal Ligament Stem Cells from Donors and Multiple Sclerosis Patients: A Comparative Study.

Multiple sclerosis (MS) is the most prevalent and progressive autoimmune disease that affects the central nervous system, and currently, no drug is available for the treatment. Stem cell therapy has received substantial attention in MS treatment. Recently, we demonstrated the immunosuppressive effects of mesenchymal stem cells derived from neural crest-originated human periodontal ligament tissue (hPDLSCs) in an in vivo model of MS. In the present study, we comparatively investigated the stemness properties of hPDLSCs derived from healthy donors and relapsing-remitting MS patients. Stem cell marker expression, cell proliferation, and differentiation capacity were studied. We found that both donor- and MS patient-derived hPDLSCs at early passage 2 showed similar expression of surface antigen markers and cell proliferation rate. Significant level of osteogenic, adipogenic, chondrogenic, and neurogenic differentiation capacities was observed in both donor- and MS patient-derived hPDLSCs. Interestingly, these cells maintained the stemness properties even at late passage 15. Senescence markers p16 and p21 expression was considerably enhanced in passage 15. Our results propose that hPDLSCs may serve as simple and potential autologous stem cell niche, which may help in personalized stem cell therapy for MS patients.

Immobilization and delivery of biologically active Lipoxin A4 using electrospinning technology.

Lipoxin (LX)A4 is a lipoxygenase-formed arachidonic acid metabolite with potent anti-inflammatory, pro-resolution properties. Its therapeutic efficacy has been largely demonstrated in a variety of cellular, preclinical and clinical models. Among these, periodontal disease, where LXA4 promotes tissue repair, also by modulating functions of human periodontal ligament stem cells (hPDLSCs). As medicated biomembranes may be particularly useful in clinical settings, where local stimulation of tissue repair is needed, we used electrospinning to embed LXA4 in membranes made of poly(ethylene oxide) (PEO) and poly(d,l-lactide) (PDLLA). These membranes were fully characterized by scanning electron microscopy, differential scanning calorimetry and biocompatibility with hPDLSCs. Here, we report that LXA4 is retained in these membranes and that ∼15-20% of the total LXA4 amount added to the reaction can be eluted from the membranes using an aqueous buffered medium. The eluted LXA4 fully retained its capability to stimulate hPDLSC proliferation. A similar effect was obtained by adding directly the LXA4-containing membranes to cells. These results demonstrate for the first time that LXA4 can be incorporated into biomembranes, which may be useful to combat local inflammation and promote tissue repair in selected clinical settings.

Nuclear translocation of PKCα isoenzyme is involved in neurogenic commitment of human neural crest-derived periodontal ligament stem cells.

Stem cells isolated from human adult tissue niche represent a promising source for neural differentiation. Human Periodontal Ligament Stem Cells (hPDLSCs) originating from the neural crest are particularly suitable for induction of neural commitment. In this study, under xeno-free culture conditions, in undifferentiated hPDLSCs and in hPDLSCs induced to neuronal differentiation by basic Fibroblast Growth Factor, the level of some neural markers have been analyzed. The hPDLSCs spontaneously express Nestin, a neural progenitor marker. In these cells, the neurogenic process induced to rearrange the cytoskeleton, form neurospheres and express higher levels of Nestin and Tyrosine Hydroxylase, indicating neural induction. Protein Kinase C (PKC) is highly expressed in neural tissue and has a key role in neuronal functions. In particular the Ca(2+) and diacylglycerol-dependent activation of PKCα isozyme is involved in the regulation of neuronal differentiation. Another main component of the pathways controlling neuronal differentiation is the Growth Associated Protein-43 (GAP-43), whose activity is strictly regulated by PKC. The aim of this study is to investigate the role of PKCα/GAP-43 nuclear signal transduction pathway during neuronal commitment of hPDLSCs. During hPDLSCs neurogenic commitment the levels of p-PKC and p-GAP-43 increased both in cytoplasmic and nuclear compartment. PKCα nuclear translocation induced GAP-43 movement to the cytoplasm, where it is known to regulate growth cone dynamics and neuronal differentiation. Moreover, the degree of cytosolic Ca(2+) mobilization appeared to be more pronounced in differentiated hPDLSCs than in undifferentiated cells. This study provides evidences of a new PKCα/GAP-43 nuclear signalling pathway that controls neuronal differentiation in hPDLSCs, leading the way to a potential use of these cells in cell-based therapy in neurodegenerative diseases.

Assessment of an efficient xeno-free culture system of human periodontal ligament stem cells.

The possibility of transplanting adult stem cells into damaged organs has opened new prospects for the treatment of several human pathologies. The purpose of this study was to develop a culture system for the expansion and production of human Periodontal Ligament Stem Cells (hPDLSCs) using a new xeno-free media formulation and ensuring the maintenance of the stem cells features comprising the multiple passage expansion, mesengenic lineage differentiation, cellular phenotype, and genomic stability, essential elements for conforming to translation to cell therapy. Somatic stem cells were isolated from the human periodontium using a minimally invasive periodontal access flap surgery in healthy donors. Expanded hPDLSCs in a xeno-free culture showed the morphological features of stem cells, expressed the markers associated with pluripotency, and a normal karyotype. Under appropriate culture conditions, hPDLSCs presented adipogenic and osteogenic potential; indeed, a very high accumulation of lipid droplets was evident in the cytoplasm of adipogenic-induced cells, and indisputable evidence of osteogenic differentiation, investigated by transmission electron microscopy, and analyzed for gene expression analysis has been shown. Based on these data, the novel xeno-free culture method might provide the basis for Good Manufacturing Procedure culture of autologous stem cells, readily accessible from human periodontium, and can be a resource to facilitate their use in human clinical studies for potential therapeutic regeneration.