Effect of Titanium and Zirconia Nanoparticles on Human Gingival Mesenchymal Stromal Cells

Nano- and microparticles are currently being discussed as potential risk factors for peri-implant disease. In the present study, we compared the responses of human gingival mesenchymal stromal cells (hG-MSCs) on titanium and zirconia nanoparticles (<100 nm) in the absence and presence of Porphyromonas gingivalis lipopolysaccharide (LPS). The primary hG-MSCs were treated with titanium and zirconia nanoparticles in concentrations up to 2.000 µg/mL for 24 h, 72 h, and 168 h. Additionally, the cells were treated with different nanoparticles (25–100 µg/mL) in the presence of P. gingivalis LPS for 24 h. The cell proliferation and viability assay and live–dead and focal adhesion stainings were performed, and the expression levels of interleukin (IL)-6, IL-8, and monocyte chemoattractant protein (MCP)-1 were measured. The cell proliferation and viability were inhibited by the titanium (>1000 µg/mL) but not the zirconia nanoparticles, which was accompanied by enhanced apoptosis. Both types of nanoparticles (>25 µg/mL) induced the significant expression of IL-8 in gingival MSCs, and a slightly higher effect was observed for titanium nanoparticles. Both nanoparticles substantially enhanced the P. gingivalis LPS-induced IL-8 production; a higher effect was observed for zirconia nanoparticles. The production of inflammatory mediators by hG-MSCs is affected by the nanoparticles. This effect depends on the nanoparticle material and the presence of inflammatory stimuli.     

A Narrative Review: Gingival Stem Cells as a Limitless Reservoir for Regenerative Medicine

The gingival tissue can be collected in an easy way and represent an accessible source to isolate gingival-derived mesenchymal stem cells (GMSCs). GMSCs are a subpopulation of dental-derived mesenchymal stem cells that show the mesenchymal stem cells (MSCs) features, such as differentiation abilities and immunomodulatory properties. Dental-derived stem cells are also expandable in vitro with genomic stability and the possibility to maintain the stemness properties over a prolonged period of passages. Moreover, several preclinical studies have documented that the extracellular vesicles (EVs) released from GMSCs possess similar biological functions and therapeutic effects. The EVs may represent a promising tool in the cell-free regenerative therapy approach. The present review paper summarized the GMSCs, their multi-lineage differentiation capacities, immunomodulatory features, and the potential use in the treatment of several diseases in order to stimulate tissue regeneration. GMSCs should be considered a good stem cell source for potential applications in tissue engineering and regenerative dentistry.     

Chitosan Hydrogel Supplemented with Metformin Promotes Neuron–like Cell Differentiation of Gingival Mesenchymal Stem Cells

Human gingival mesenchymal stem cells (GMSCs) are derived from migratory neural crest stem cells and have the potential to differentiate into neurons. Metformin can inhibit stem–cell aging and promotes the regeneration and development of neurons. In this study, we investigated the potential of metformin as an enhancer on neuronal differentiation of GMSCs in the growth environment of chitosan hydrogel. The crosslinked chitosan/β–glycerophosphate hydrogel can form a perforated microporous structure that is suitable for cell growth and channels to transport water and macromolecules. GMSCs have powerful osteogenic, adipogenic and chondrogenic abilities in the induction medium supplemented with metformin. After induction in an induction medium supplemented with metformin, Western blot and immunofluorescence results showed that GMSCs differentiated into neuron–like cells with a significantly enhanced expression of neuro–related markers, including Nestin (NES) and β–Tubulin (TUJ1). Proteomics was used to construct protein profiles in neural differentiation, and the results showed that chitosan hydrogels containing metformin promoted the upregulation of neural regeneration–related proteins, including ATP5F1, ATP5J, NADH dehydrogenase (ubiquinone) Fe–S protein 3 (NDUFS3), and Glutamate Dehydrogenase 1 (GLUD1). Our results help to promote the clinical application of stem–cell neural regeneration.     

Engineering a 3D In Vitro Model of Human Gingival Tissue Equivalent with Genipin/Cytochalasin D

Although three-dimensional (3D) co-culture of gingival keratinocytes and fibroblasts-populated collagen gel can mimic 3D structure of in vivo tissue, the uncontrolled contraction of collagen gel restricts its application in clinical and experimental practices. We here established a stable 3D gingival tissue equivalent (GTE) using hTERT-immortalized gingival fibroblasts (hGFBs)-populated collagen gel directly crosslinked with genipin/cytochalasin D and seeding hTERT-immortalized gingival keratinocytes (TIGKs) on the upper surface for a 2-week air–liquid interface co-culture. MTT assay was used to measure the cell viability of GTEs. GTE size was monitored following culture period, and the contraction was analyzed. Immunohistochemical assay was used to analyze GTE structure. qRT-PCR was conducted to examine the mRNA expression of keratinocyte-specific genes. Fifty µM genipin (G50) or combination (G + C) of G50 and 100 nM cytochalasin D significantly inhibited GTE contraction. Additionally, a higher cell viability appeared in GTEs crosslinked with G50 or G + C. GTEs crosslinked with genipin/cytochalasin D showed a distinct multilayered stratified epithelium that expressed keratinocyte-specific genes similar to native gingiva. Collagen directly crosslinked with G50 or G + C significantly reduced GTE contraction without damaging the epithelium. In summary, the TIGKs and hGFBs can successfully form organotypic multilayered cultures, which can be a valuable tool in the research regarding periodontal disease as well as oral mucosa disease. We conclude that genipin is a promising crosslinker with the ability to reduce collagen contraction while maintaining normal cell function in collagen-based oral tissue engineering.     

Cell Therapy Based on Gingiva-Derived Mesenchymal Stem Cells Seeded in a Xenogeneic Collagen Matrix for Root Coverage of RT1 Gingival Lesions: An In Vivo Experimental Study

(1) Background: To investigate the effect of a xenogeneic collagen matrix (CMX) seeded with autologous gingiva-derived mesenchymal cells (GMSCs) when combined with a coronally advanced flap (CAF) in the treatment of localized gingival recession type 1 (RT1). (2) Methods: Dehiscence-type defects were created in seven dogs. GMSCs were isolated, transfected with a vector carrying green fluorescent protein (GFP) and expanded. Once chronified, the defects were randomly treated with (1) CAF plus the combination of CMX and GFP⁺ GMSCs, (2) CAF plus CMX with autologous fibroblasts, (3) CAF plus CMX and (4) CAF alone. Histological and clinical outcomes at 2- and 6-week healing periods were analyzed and compared among groups. (3) Results: Histologically, the addition of autologous cells to the CMX resulted in reduced inflammation and a variable degree of new cementum/bone formation. CMX plus GMSCs resulted in greater mean recession reduction (1.42; SD = 1.88 mm) and percentage of teeth with recession reduction of ≥2 mm (57%) when compared to the other groups, although these differences were not statistically significant. (4) Conclusions: The histometric and clinical results indicated a positive trend favouring the combination of CMX and GMSCs with the CAF when compared to the groups without cells, although these differences were not statistically significant.     

Evaluation of Platelet-Derived Extracellular Vesicles in Gingival Fibroblasts and Keratinocytes for Periodontal Applications

Gingival regeneration aims at restoring the architecture and functionality of oral damaged tissue. Different biomaterials or biological materials have been tested for tissue repair, such as platelet concentrates such as PL. In this article, the use of extracellular vesicles (EVs) derived from platelet lysate (PL) and their combination with hyaluronic acid biomaterials (HA) in an in vitro wound healing assay is investigated. EVs were isolated by size exclusion chromatography from PL. In addition, HA gels were formulated with PL or EVs. EVs or HA combined with EVs (HA-EVs) were tested in vitro in gingival fibroblasts and keratinocytes for biocompatibility (LDH activity and metabolic activity) and by an in vitro wound-healing assay and gene expression analysis. EVs and EVs-HA treatments were biocompatible in gingival fibroblasts and keratinocytes and showed an increase in wound healing in vitro compared to control. Moreover, changes in gene expression related to extracellular matrix remodeling were observed after the treatment with EVs. EVs can be combined with HA biomaterials, showing good biocompatibility and preserving their activity and functionality. Therefore, platelet-derived EVs could emerge as a new application for periodontal regeneration in combination with biomaterials in order to enhance their clinical use.     

A New Human Platelet Lysate for Mesenchymal Stem Cell Production Compliant with Good Manufacturing Practice Conditions

Mesenchymal stem cells (MSCs) are classified as advanced therapy medicinal products, a new category of GMP (good manufacturing practice)-compliant medicines for clinical use. We isolated MSCs from 5 bone marrow (BM) samples using human platelet lysate (HPL) instead of foetal bovine serum (FBS). We used a new method of HPL production consisting of treating platelet (PLTs) pools with Ca-Gluconate to form a gel clot, then mechanically squeezing to release growth factors. We compared the new HPL (HPL-S) with the standard (HPL-E) obtained by freezing/thawing cycles and by adding heparin. HPL-S had not PLTs and fibrinogen but the quantity of proteins and growth factors was comparable to HPL-E. Therefore, HPL-S needed fewer production steps to be in compliance with GMP conditions. The number of colonies forming unit-fibroblasts (CFU-F) and the maintenance of stem markers showed no significant differences between MSCs with HPL-E and HPL-S. The cumulative population doubling was higher in MSCs with HPL-E in the earlier passages, but we observed an inverted trend of cell growth at the fourth passage. Immunophenotypic analysis showed a significant lower expression of HLA-DR in the MSCs with HPL-S (1.30%) than HPL-E (14.10%). In conclusion, we demonstrated that HPL-S is an effective alternative for MSC production under GMP conditions.     

Rapid Production and Genetic Stability of Human Mesenchymal Progenitor Cells Derived from Human Somatic Cell Nuclear Transfer-Derived Pluripotent Stem Cells

Pluripotent stem cell-derived mesenchymal progenitor cells (PSC-MPCs) are primarily derived through two main methods: three-dimensional (3D) embryoid body-platform (EB formation) and the 2D direct differentiation method. We recently established somatic cell nuclear transfer (SCNT)-PSC lines and showed their stemness. In the present study, we produced SCNT-PSC-MPCs using a novel direct differentiation method, and the characteristics, gene expression, and genetic stability of these MPCs were compared with those derived through EB formation. The recovery and purification of SCNT-PSC-Direct-MPCs were significantly accelerated compared to those of the SCNT-PSC-EB-MPCs, but both types of MPCs expressed typical surface markers and exhibited similar proliferation and differentiation potentials. Additionally, the analysis of gene expression patterns using microarrays showed very similar patterns. Moreover, array CGH analysis showed that both SCNT-PSC-Direct-MPCs and SCNT-PSC-EB-MPCs exhibited no significant differences in copy number variation (CNV) or single-nucleotide polymorphism (SNP) frequency. These results indicate that SCNT-PSC-Direct-MPCs exhibited high genetic stability even after rapid differentiation into MPCs, and the rate at which directly derived MPCs reached a sufficient number was higher than that of MPCs derived through the EB method. Therefore, we suggest that the direct method of differentiating MPCs from SCNT-PSCs can improve the efficacy of SCNT-PSCs applied to allogeneic transplantation.     

RNA Sequencing Reveals the Upregulation of FOXO Signaling Pathway in Porphyromonas gingivalis Persister-Treated Human Gingival Epithelial Cells

Porphyromonas gingivalis as the keystone periodontopathogen plays a critical role in the pathogenesis of periodontitis, and crucially accounts for inflammatory comorbidities such as cardiovascular disease and Alzheimer′s disease. We recently identified the existence of P. gingivalis persisters and revealed the unforeseen perturbation of innate response in human gingival epithelial cells (HGECs) due to these noxious persisters. Herein, RNA sequencing revealed how P. gingivalis persisters affected the expression profile of cytokine genes and related signaling pathways in HGECs. Results showed that metronidazole-treated P. gingivalis persisters (M-PgPs) impaired the innate host defense of HGECs, in a similar fashion to P. gingivalis. Notably, over one thousand differentially expressed genes were identified in HGECs treated with M-PgPs or P. gingivalis with reference to the controls. Gene Ontology and KEGG pathway analysis demonstrated significantly enriched signaling pathways, such as FOXO. Importantly, the FOXO1 inhibitor rescued the M-PgP-induced disruption of cytokine expression. This study suggests that P. gingivalis persisters may perturb innate host defense, through the upregulation of the FOXO signaling pathway. Thus, the current findings could contribute to developing new approaches to tackling P. gingivalis persisters for the effective control of periodontitis and P. gingivalis-related inflammatory comorbidities.     

Injectable Human Hair Keratin–Fibrinogen Hydrogels for Engineering 3D Microenvironments to Accelerate Oral Tissue Regeneration

Traumatic injury of the oral cavity is atypical and often accompanied by uncontrolled bleeding and inflammation. Injectable hydrogels have been considered to be promising candidates for the treatment of oral injuries because of their simple formulation, minimally invasive application technique, and site-specific delivery. Fibrinogen-based hydrogels have been widely explored as effective materials for wound healing in tissue engineering due to their uniqueness. Recently, an injectable foam has taken the spotlight. However, the fibrin component of this biomaterial is relatively stiff. To address these challenges, we created keratin-conjugated fibrinogen (KRT-FIB). This study aimed to develop a novel keratin biomaterial and assess cell–biomaterial interactions. Consequently, a novel injectable KRT-FIB hydrogel was optimized through rheological measurements, and its injection performance, swelling behavior, and surface morphology were investigated. We observed an excellent cell viability, proliferation, and migration/cell–cell interaction, indicating that the novel KRT-FIB-injectable hydrogel is a promising platform for oral tissue regeneration with a high clinical applicability.     

Therapeutic Potential of Human Stem Cell Implantation in Alzheimer’s Disease

Alzheimer’s disease (AD) is a progressive debilitating neurodegenerative disease and the most common form of dementia in the older population. At present, there is no definitive effective treatment for AD. Therefore, researchers are now looking at stem cell therapy as a possible treatment for AD, but whether stem cells are safe and effective in humans is still not clear. In this narrative review, we discuss both preclinical studies and clinical trials on the therapeutic potential of human stem cells in AD. Preclinical studies have successfully differentiated stem cells into neurons in vitro, indicating the potential viability of stem cell therapy in neurodegenerative diseases. Preclinical studies have also shown that stem cell therapy is safe and effective in improving cognitive performance in animal models, as demonstrated in the Morris water maze test and novel object recognition test. Although few clinical trials have been completed and many trials are still in phase I and II, the initial results confirm the outcomes of the preclinical studies. However, limitations like rejection, tumorigenicity, and ethical issues are still barriers to the advancement of stem cell therapy. In conclusion, the use of stem cells in the treatment of AD shows promise in terms of effectiveness and safety.     

Quality Control of Human Pluripotent Stem Cell Colonies by Computational Image Analysis Using Convolutional Neural Networks

Human pluripotent stem cells are promising for a wide range of research and therapeutic purposes. Their maintenance in culture requires the deep control of their pluripotent and clonal status. A non-invasive method for such control involves day-to-day observation of the morphological changes, along with imaging colonies, with the subsequent automatic assessment of colony phenotype using image analysis by machine learning methods. We developed a classifier using a convolutional neural network and applied it to discriminate between images of human embryonic stem cell (hESC) colonies with “good” and “bad” morphological phenotypes associated with a high and low potential for pluripotency and clonality maintenance, respectively. The training dataset included the phase-contrast images of hESC line H9, in which the morphological phenotype of each colony was assessed through visual analysis. The classifier showed a high level of accuracy (89%) in phenotype prediction. By training the classifier on cropped images of various sizes, we showed that the spatial scale of ~144 μm was the most informative in terms of classification quality, which was an intermediate size between the characteristic diameters of a single cell (~15 μm) and the entire colony (~540 μm). We additionally performed a proteomic analysis of several H9 cell samples used in the computational analysis and showed that cells of different phenotypes differentiated at the molecular level. Our results indicated that the proposed approach could be used as an effective method of non-invasive automated analysis to identify undesirable developmental anomalies during the propagation of pluripotent stem cells.     

Human Pluripotent Stem Cell-Derived Neural Progenitor Cells Promote Retinal Ganglion Cell Survival and Axon Recovery in an Optic Nerve Compression Animal Model

Human pluripotent stem cell-derived neural progenitor cells (NPCs) have the potential to recover from nerve injury. We previously reported that human placenta-derived mesenchymal stem cells (PSCs) have neuroprotective effects. To evaluate the potential benefit of NPCs, we compared them to PSCs using R28 cells under hypoxic conditions and a rat model of optic nerve injury. NPCs and PSCs (2 × 106 cells) were injected into the subtenon space. After 1, 2, and 4 weeks, we examined changes in target proteins in the retina and optic nerve. NPCs significantly induced vascular endothelial growth factor (Vegf) compared to age-matched shams and PSC groups at 2 weeks; they also induced neurofilaments in the retina compared to the sham group at 4 weeks. In addition, the expression of brain-derived neurotrophic factor (Bdnf) was high in the retina in the NPC group at 2 weeks, while expression in the optic nerve was high in both the NPC and PSC groups. The low expression of ionized calcium-binding adapter molecule 1 (Iba1) in the retina had recovered at 2 weeks after NPC injection and at 4 weeks after PSC injection. The expression of the inflammatory protein NLR family, pyrin domain containing 3 (Nlrp3) was significantly reduced at 1 week, and that of tumor necrosis factor-α (Tnf-α) in the optic nerves of the NPC group was lower at 2 weeks. Regarding retinal ganglion cells, the expressions of Brn3a and Tuj1 in the retina were enhanced in the NPC group compared to sham controls at 4 weeks. NPC injections increased Gap43 expression from 2 weeks and reduced Iba1 expression in the optic nerves during the recovery period. In addition, R28 cells exposed to hypoxic conditions showed increased cell survival when cocultured with NPCs compared to PSCs. Both Wnt/β-catenin signaling and increased Nf-ĸb could contribute to the rescue of damaged retinal ganglion cells via upregulation of neuroprotective factors, microglial engagement, and anti-inflammatory regulation by NPCs. This study suggests that NPCs could be useful for the cellular treatment of various optic neuropathies, together with cell therapy using mesenchymal stem cells.     

A Novel Cell-Based Model for a Rare Disease: The Tks4-KO Human Embryonic Stem Cell Line as a Frank-Ter Haar Syndrome Model System

Tyrosine kinase substrate with four SH3 domains (Tks4) scaffold protein plays roles in cell migration and podosome formation and regulates systemic mechanisms such as adult bone homeostasis and adipogenesis. Mutations in the Tks4 gene (SH3PXD2b) cause a rare developmental disorder called Frank-Ter Haar syndrome (FTHS), which leads to heart abnormalities, bone tissue defects, and reduced adiposity. We aimed to produce a human stem cell-based in vitro FTHS model system to study the effects of the loss of the Tks4 protein in different cell lineages and the accompanying effects on the cell signalome. To this end, we used CRISPR/Cas9 (clustered, regularly interspaced, short palindromic repeats (CRISPR)/CRISPR associated (Cas9)) to knock out the SH3PXD2b gene in the HUES9 human embryonic stem cell line (hESC), and we obtained stable homo- and heterozygous knock out clones for use in studying the potential regulatory roles of Tks4 protein in embryonic stem cell biology. Based on pluripotency marker measurements and spontaneous differentiation capacity assays, we concluded that the newly generated Tks4-KO HUES9 cells retained their embryonic stem cell characteristics. We propose that the Tks4-KO HUES9 cells could serve as a tool for further cell differentiation studies to investigate the involvement of Tks4 in the complex disorder FTHS. Moreover, we successfully differentiated all of the clones into mesenchymal stem cells (MSCs). The derived MSC cultures showed mesenchymal morphology and expressed MSC markers, although the expression levels of mesodermal and osteogenic marker genes were reduced, and several EMT (epithelial mesenchymal transition)-related features were altered in the Tks4-KO MSCs. Our results suggest that the loss of Tks4 leads to FTHS by altering cell lineage differentiation and cell maturation processes, rather than by regulating embryonic stem cell potential.     

The Long-Term Culture of Human Fibroblasts Reveals a Spectroscopic Signature of Senescence

Aging is a complex process which leads to progressive loss of fitness/capability/ability, increasing susceptibility to disease and, ultimately, death. Regardless of the organism, there are some features common to aging, namely, the loss of proteostasis and cell senescence. Mammalian cell lines have been used as models to study the aging process, in particular, cell senescence. Thus, the aim of this study was to characterize the senescence-associated metabolic profile of a long-term culture of human fibroblasts using Fourier Transform Infrared and Nuclear Magnetic Resonance spectroscopy. We sub-cultivated fibroblasts from a newborn donor from passage 4 to passage 17 and the results showed deep changes in the spectroscopic profile of cells over time. Late passage cells were characterized by a decrease in the length of fatty acid chains, triglycerides and cholesterol and an increase in lipid unsaturation. We also found an increase in the content of intermolecular β-sheets, possibly indicating an increase in protein aggregation levels in cells of later passages. Metabolic profiling by NMR showed increased levels of extracellular lactate, phosphocholine and glycine in cells at later passages. This study suggests that spectroscopy approaches can be successfully used to study changes concomitant with cell senescence and validate the use of human fibroblasts as a model to monitor the aging process.     

The Effects of Synthetic Oligopeptide Derived from Enamel Matrix Derivative on Cell Proliferation and Osteoblastic Differentiation of Human Mesenchymal Stem Cells

Enamel matrix derivative (EMD) is widely used in periodontal tissue regeneration therapy. However, because the bioactivity of EMD varies from batch to batch, and the use of a synthetic peptide could avoid use from an animal source, a completely synthetic peptide (SP) containing the active component of EMD would be useful. In this study an oligopeptide synthesized derived from EMD was evaluated for whether it contributes to periodontal tissue regeneration. We investigated the effects of the SP on cell proliferation and osteoblast differentiation of human mesenchymal stem cells (MSCs), which are involved in tissue regeneration. MSCs were treated with SP (0 to 1000 ng/mL), to determine the optimal concentration. We examined the effects of SP on cell proliferation and osteoblastic differentiation indicators such as alkaline phosphatase activity, the production of procollagen type 1 C-peptide and osteocalcin, and on mineralization. Additionally, we investigated the role of extracellular signal-related kinases (ERK) in cell proliferation and osteoblastic differentiation induced by SP. Our results suggest that SP promotes these processes in human MSCs, and that ERK inhibitors suppress these effects. In conclusion, SP promotes cell proliferation and osteoblastic differentiation of human MSCs, probably through the ERK pathway.     

A New Human Platelet Lysate for Mesenchymal Stem Cell Production Compliant with Good Manufacturing Practice Conditions Preserves the Chemical Characteristics and Biological Activity of Lyo-Secretome Isolated by Ultrafiltration

Recently, we proposed a Good Manufacturing Practice (GMP)-compliant production process for freeze-dried mesenchymal stem cell (MSC)-secretome (lyo-secretome): after serum starvation, the cell supernatant was collected, and the secretome was concentrated by ultrafiltration and freeze-dried, obtaining a standardized ready-to-use and stable powder. In this work, we modified the type of human platelet lysate (HPL) used as an MSC culture supplement during the lyo-secretome production process: the aim was to verify whether this change had an impact on product quality and also whether this new procedure could be validated according to GMP, proving the process robustness. MSCs were cultured with two HPLs: the standard previously validated one (HPL-E) and the new one (HPL-S). From the same pool of platelets, two batches of HPL were obtained: HPL-E (by repeated freezing and thawing cycles) and HPL-S (by adding Ca-gluconate to form a clot and its subsequent mechanical wringing). Bone marrow MSCs from three donors were separately cultured with the two HPLs until the third passage and then employed to produce lyo-secretome. The following indicators were selected to evaluate the process performance: (i) the lyo-secretome quantitative composition (in lipids and proteins), (ii) the EVs size distribution, and (iii) anti-elastase and (iv) immunomodulant activity as potency tests. The new HPL supplementation for MSCs culture induced only a few minimal changes in protein/lipid content and EVs size distribution; despite this, it did not significantly influence biological activity. The donor intrinsic MSCs variability in secretome secretion instead strongly affected the quality of the finished product and could be mitigated by concentrating the final product to reach a determined protein (and lipid) concentration. In conclusion, the modification of the type of HPL in the MSCs culture during lyo-secretome production induces only minimal changes in the composition but not in the potency, and therefore, the new procedure can be validated according to GMP.