Human Pulp Stem Cells Research Articles

Histone Deacetylase Inhibitors Restore the Odontogenic Differentiation Potential of Dental Pulp Stem Cells under Hyperglycemic Conditions.

Complications arising from diabetes can result in stem cell dysfunction, impairing their ability to undergo differentiation into various cellular lineages. The present study evaluated the effect of histone deacetylase inhibitors, Valproic acid and Trichostatin A, on the odontogenic differentiation potential of dental pulp stem cells under hyperglycemic conditions. Streptozotocin (STZ) induced diabetes mellitus in 12 male Wistar rats. Dental parameters were examined using micro-computed tomography. The odontogenic potential of human pulp stem cells exposed to 30 mM glucose was assessed through alkaline phosphatase assays, examination of gene expression for dentin matrix protein 1 and dentin sialoprotein using real-time PCR, and alizarin red staining for calcium deposition. Along with reduced dentin thickness and root length in diabetic rats, the results revealed a significant increase in histone deacetylase 3 and 2 gene expressions in isolated diabetic pulp tissues compared to the control groups. The gene expression of odontogenic-related markers and alkaline phosphatase activity in human cultured pulp stem cells under hyperglycemic conditions significantly decreased. Adding Valproic acid and Trichostatin A restored the odontogenic differentiation markers, including calcium deposition, gene expression of dentin sialophosphoprotein, dentin matrix protein 1, and alkaline phosphatase activity. The data suggests that hyperglycemic conditions negatively impact the odontogenic potential of pulp mesenchymal stem cells. However, histone deacetylase inhibitors improve the impaired odontogenic differentiation capacity. This study implies that histone deacetylases may represent a potential therapeutic target for enhancing the regenerative mineralization of pulp cells in diabetic patients.

Evaluation of in vitro biocompatibility of human pulp stem cells with allogeneic, alloplastic, and xenogeneic grafts under the influence of extracellular vesicles

Therapies using dental pulp stem cells (DPSCs) or stem cell-derived extracellular vesicles (EVs) have shown promising applications for bone tissue engineering. This in vitro experiment evaluated the joint osteogenic capability of DPSCs and EVs on alloplastic (maxresorp), allogeneic (maxgraft), and xenogeneic (cerabone) bone grafts. We hypothesize that osteogenic differentiation and the proliferation of human DPSCs vary between bone grafts and are favorable under the influence of EVs. DPSCs were obtained from human wisdom teeth, and EVs derived from DPSCs were isolated from cell culture medium. DPSCs were seeded on alloplastic, allogeneic, and xenogeneic bone graft substitutes for control, and the same scaffolds were administered with EVs in further groups. The cellular uptake of EVs into DPSC cells was assessed by confocal laser scanning microscopy. Cell vitality staining and calcein acetoxymethyl ester staining were used to evaluate cell attachment and proliferation. Cell morphology was determined using scanning electron microscopy, and osteogenic differentiation was explored by alkaline phosphatase and Alizarin red staining. Within the limitations of an in vitro study without pathologies, the results suggest that especially the use of xenogeneic bone graft substitutes with DPSCS and EVs may represent a promising treatment approach for alveolar bone defects.

Pulp-Dentin Complex Regeneration with Cell Transplantation Technique Using Stem Cells Derived from Human Deciduous Teeth: Histological and Immunohistochemical Study in Immunosuppressed Rats.

The aim of this study was to histologically verify the performance of pulp-derived stem cells used in the pulp-dentin complex regeneration. Maxillary molars of 12 immunosuppressed rats were divided into two groups: the SC (stem cells) group, and the PBS (just standard phosphate-buffered saline) group. After pulpectomy and canal preparation, the teeth received the designated materials, and the cavities were sealed. After 12 weeks, the animals were euthanized, and the specimens underwent histological processing and qualitative evaluation of intracanal connective tissue, odontoblast-like cells, intracanal mineralized tissue, and periapical inflammatory infiltrate. Immunohistochemical evaluation was performed to detect dentin matrix protein 1 (DMP1). In the PBS group, an amorphous substance and remnants of mineralized tissue were observed throughout the canal, and abundant inflammatory cells were observed in the periapical region. In the SC group, an amorphous substance and remnants of mineralized tissue were observed throughout the canal; odontoblasts-like cells immunopositive for DMP1 and mineral plug were observed in the apical region of the canal; and a mild inflammatory infiltrate, intense vascularization, and neoformation of organized connective tissue were observed in the periapical region. In conclusion, the transplantation of human pulp stem cells promoted partial pulp tissue neoformation in adult rat molars.

Dental stem cells and tissue regeneration in odontology: a brief systematic review

Introduction: New ideas for tooth and tissue regeneration began to appear with rapid developments in tissue engineering theories and technologies. Numerous types of stem cells have been isolated from dental tissue, such as dental pulp stem cells (DPSC), human pulp stem cells isolated from exfoliated primary teeth (SHED), periodontal ligament stem cells (PDLSC), apical papillary stem (SCAP) and dental follicular cells (DFC). All these cells can regenerate tooth tissue. Objective: It was to present the main considerations of bioengineering techniques and report the results obtained in experiments with dental stem cells, as well as their real trends in application in dentistry. Methods: The systematic review rules of the PRISMA Platform were followed. The search was carried out from October to December 2022 in the Scopus, PubMed, Science Direct, Scielo, and Google Scholar databases, using articles from 2001 to 2022. The quality of the studies was based on the GRADE instrument and the risk of bias was analyzed accordingly, according to the Cochrane instrument. Results and Conclusion: A total of 118 articles were found, 27 articles were evaluated and 16 were included and developed in this systematic review study. Considering the Cochrane tool for risk of bias, the overall assessment resulted in 29 studies with a high risk of bias and 42 studies that did not meet GRADE. It is concluded that the collection of mesenchymal stem cells from deciduous teeth should be widely disseminated, as it is of great importance and wide applicability, allowing the repair of the most diverse cell types. In addition, it has several advantages, such as a non-invasive technique, respecting the period of dentition change, there are more than 20 collection possibilities, it presents high compatibility with the donor and family members and storage for an indefinite period. The studies showed that implantation of stem cells from deciduous teeth led to the regeneration of three-dimensional pulp tissue equipped with blood vessels and sensory nerves up to 12 months after treatment, as well as increased root length and reduced apical foramen width, not showing adverse events over 24 months of follow-up.

In vitro comparison of the osteogenic capability of human pulp stem cells on alloplastic, allogeneic, and xenogeneic bone scaffolds

BackgroundA rigorous search for alternatives to autogenous bone grafts to avoid invasiveness at the donor site in the treatment of maxillomandibular bone defects. Researchers have used alloplastic, allogeneic, and xenogeneic bone graft substitutes in clinical studies with varying degrees of success, although their in vitro effects on stem cells remain unclear. Dental pulp stem cells (DPSCs) can potentially enhance the bone regeneration of bone graft substitutes. The present in vitro study investigates the osteogenic capability of DPSCs on alloplastic (biphasic calcium phosphate [BCP]), allogeneic (freeze-dried bone allografts [FDBAs]), and xenogeneic (deproteinized bovine bone mineral [DBBM]) bone grafts.MethodsHuman DPSCs were seeded on 0.5 mg/ml, 1 mg/ml, and 2 mg/ml of BCP, FDBA, and DBBM to evaluate the optimal cell growth and cytotoxicity. Scaffolds and cell morphologies were analyzed by scanning electron microscopy (SEM). Calcein AM and cytoskeleton staining were performed to determine cell attachment and proliferation. Alkaline phosphatase (ALP) and osteogenesis-related genes expressions was used to investigate initial osteogenic differentiation.ResultsCytotoxicity assays showed that most viable DPSCs were present at a scaffold concentration of 0.5 mg/ml. The DPSCs on the DBBM scaffold demonstrated a significantly higher proliferation rate of 214.25 ± 16.17 (p < 0.001) cells, enhancing ALP activity level and upregulating of osteogenesis-related genes compared with other two scaffolds.ConclusionDBBP scaffold led to extremely high cell viability, but also promoted proliferation, attachment, and enhanced the osteogenic differentiation capacity of DPSCs, which hold great potential for bone regeneration treatment; however, further studies are necessary.

Bioenergetics of photobiomodulated osteoblast mitochondrial cells derived from human pulp stem cells: systematic review.

Dental pulp cells are a source of multipotent mesenchymal stem cells with a high proliferation rate and multilineage differentiation potential. This study investigated the photobiomodulated bioenergetic effects of mitochondria in osteoblasts that differentiated from human pulp stem cells. The systematic review followed PRISMA guidelines. The PICO question was formulated. Criteria for inclusion and exclusion were established prior to searches being performed on the PubMed/MEDLINE, Embase, and Scopus. Articles were identified and included if published in English within last 10years; photobiomodulation or low-level laser therapy were discussed; the delivery parameters for dose and time were included and the studies focused on bioenergetics of osteoblast mitochondria. Studies excluded were non-human dental pulp tissue and in vivo studies. A total number of 110 articles were collated, 106 were excluded leaving a total of 4 articles. These studies demonstrated that in vitro use of photobiomodulation was performed using different laser and LED types; InGaAlP; InGaN; and InGaAsP with average wavelengths of 630 to 940nm. Primary human osteoblastic STRO-1 and mesenchymal stem cell lineages were studied. Three out of four articles confirmed positive bioenergetic effects of photobiomodulation on mitochondria of osteoblasts derived from human pulp cells. This systematic review demonstrated a lack of adequate reporting of bioenergetics of osteoblast mitochondria after photobiomodulation treatment.

Chitosan and stemcells: A synchrony for regeneration

Background: Stem cells have infinite self-renewing capacity and potential for differentiation and regeneration. Human dental pulp stem cells (hDPSCs) from the extracted tooth are a rich source of mesenchymal stem cells, which can regenerate into osteogenic, chondrogenic, or adipogenic lineages. Chitosan which is a porous structure may have the ability to house these stem cells for regeneration. This study was to determine the effectiveness of growth of hDPSCs in the chitosan scaffolds. Methodology: The study was conducted in the Regenerative Medicine Laboratory, Dr. D.Y. Patil Dental College and Hospital in coordination with Department of Oral and Maxillofacial Surgery, Dr. D.Y. Patil Dental College and Hospital, and Dr. D.Y. Patil Institute of Pharmaceutical Sciences and Research, Pimpri, Pune. The chitosan scaffolds were prepared at Dr. D.Y. Patil Institute of Pharmaceutical Sciences and Research. Twenty extracted human dental pulp were used for stem cells differentiation. Results: The chitosan scaffold showed an excellent growth of chondrogenic cells per unit in × 40 magnification, and hence, this sets a benchmark for hDPSC study in India. Conclusion: hDPSCs in porous chitosan scaffolds help us to regenerate chondrogenic cells and will initiate the research in an interdisciplinary approach toward cartilage regeneration.

Pulp stem cells derived from human permanent and deciduous teeth: Biological characteristics and therapeutic applications.

Human pulp stem cells (PSCs) include dental pulp stem cells (DPSCs) isolated from dental pulp tissues of human extracted permanent teeth and stem cells from human exfoliated deciduous teeth (SHED). Depending on their multipotency and sensitivity to local paracrine activity, DPSCs and SHED exert therapeutic applications at multiple levels beyond the scope of the stomatognathic system. This review is specifically concentrated on PSC‐updated biological characteristics and their promising therapeutic applications in (pre)clinical practice. Biologically, distinguished from conventional mesenchymal stem cell markers in vitro, NG2, Gli1, and Celsr1 have been evidenced as PSC markers in vivo. Both perivascular cells and glial cells account for PSC origin. Therapeutically, endodontic regeneration is where PSCs hold the most promises, attributable of PSCs' robust angiogenic, neurogenic, and odontogenic capabilities. More recently, the interplay between cell homing and liberated growth factors from dentin matrix has endowed a novel approach for pulp‐dentin complex regeneration. In addition, PSC transplantation for extraoral tissue repair and regeneration has achieved immense progress, following their multipotential differentiation and paracrine mechanism. Accordingly, PSC banking is undergoing extensively with the intent of advancing tissue engineering, disease remodeling, and (pre)clinical treatments.

Upregulation of Neuroprogenitor and Neural Markers via Enforced miR-124 and Growth Factor Treatment.

Previous studies have shown that miR-124 plays an important role in the development of auditory neurons, which are degenerated in the sensorineural hearing loss. However, whether the combined use of miR-124 and growth factors can increase the expression of neural related markers in human dental pulp stem cells has been remained unknown so far. In this study, human dental pulp stem cells were transfected with miR-124 following treatment with brain-derived neurotrophic factor or epidermal growth factor/basic fibroblast growth factor. The expression of some neural related markers (nestin, SOX2, β-tubulin III, MAP2, and peripherin) was analyzed in two groups by qRT-PCR or immunofluorescence. Cellular treatment resulted in morphological changes including neurosphere-like colonies formation. Nestin and SOX2 were up-regulated, and MAP2 and peripherin were down-regulated in dental pulp stem cells transfected by miR-124 following treatment with brain-derived neurotrophic factor. Replacement of brain-derived neurotrophic factor with epidermal growth factor/ basic fibroblast growth factor resulted in the up-regulation of nestin, MAP2, peripherin, and β-tubulin III and down-regulation of SOX2. The expression of SOX2 and nestin was also confirmed by immunofluorescence. The combination of miR-124 and growth factors would provide a promising starting point for upregulating the neural progenitor markers in human dental pulp stem cells.

Regulation of Wnt signaling pathway on calcium hydroxide-promoted osteogenic differentiation of hDPSCs

Objective To investigate the role of Wnt/β-catenin signaling pathway in the osteogenic differentiation of human dental pulp stem cells (hDPSCs). Methods After 14 days of the calcium hydroxide training, the cytoskeletal changes of hDPSCs, the expression of β-catenin, i.e. the key promoter of in the Wnt signaling pathway, and the cell localization were detected by laser scanning confocal technique. The Wnt signaling pathways were up-regulated and inhibited, and the osteogenic differentiation and mineralization of hDPSCs were detected by Western Blot and alizarin red staining after 14 days of the training. Results The cytoskeleton of hDPSCs was rearranged by the effect of calcium hydroxide, and the β-catenin migration from nucleus to cytoplasm were observed. The number of calcium nodules in hDPSCs was decreased after blocking Wnt signaling pathway by Dickkopf-related protein 1 (DKK-1). The calcium hydroxide treatment can promoted dentin sialophosphoprotein (DSPP) expression in hDPSCs. Conclusions Calcium hydroxide can down-regulate the expression of canonical Wnt signaling pathway and promote osteogenic differentiation and mineralization and odontogenetic differentiation of hDPSCs. Key words: Calcium hydroxide; Osteogenic differentiation; Human dental pulp stem; Wnt pathway

Investigation the role of Fe3O4 in the silica based bioactive polycrystalline modified with ZnO and CaF2

The aim of this article was to investigation the function of silica based polycrystalline that modified with ZnO and CaF2 and doped with 6% Fe3O4. The human pulp stem cells cultured in the presence of samples. The effects of heating-induced crystallization on the glass structure were studied by DSC and x-ray diffraction. The particle size of prepared samples was studied by TEM analysis. The bioactivity of samples were analyzed by apatite-formation ability in DMEM solution through SEM-EDX, biocompatibility was evaluated through the SHEDs cells proliferation, adhesion and spreading on surface of samples by MTT assay and ALP activity assay. Optical and SEM images displayed that co-operation of ZnO and CaF2 led to appropriate circumstance for cells proliferation, adhesion and spreading. Proliferation of pulp stem cells after 7 d of incubation in culture media containing ZC8, FC8 and ZFC4 yielded values 0.3, 1.7 and 2.8 in comparison with control, respectively.

Bioactive glass 45S5-silk fibroin membrane supports proliferation and differentiation of human dental pulp stem cells

To investigate the effect of bioactivity glass 45S5- silk fibroin(BG45S5- SF) membrane on growth, proliferation and differentiation of human dental pulp stem cells(hDPSC), and to provide new ideas and method for the regeneration of pulp-dentine complex. hDPSC seed on pure silk fibroin membrane (protein membrane group) and BG45S5-SF membrane with different concentrations(1 000, 5 000 mg/L, composite membrane group A and B, respectively) were prepared, and the materials were incubated in cell culture fluid for 24 h. No material membrane orifice plate was used as blank control group. Contact angle meter was used to measure surface contact angle of protein membrane and composite membrane group(each group had three repeated holes). Cell proliferation was assessed by cell counting kit- 8 on the 4, 7, 14, and 21 days. The state of adhesion and growth of hDPSC on the materials surface was evaluated by scanning electron microscopy and cytoskeleton staining; and alkaline phosphatase (ALP) activity was measured to evaluate the cell differentiation potential. The expression of odontoblastic differentiation-related genes was measured by real-time PCR. Surface contact angle of the protein membrane group and composite membrane group A and group B were 89.51° ± 0.12°, 70.32° ± 0.07° and 71.31° ± 0.09° respectively. hDPSC adhered well on each materials surface on the 7, 14, 21 days, ALP activity and differentiation genes of composite membrane group A and B rised more significantly than the blank control group and protein membrane group did (P<0.05). Dentin matrix protein1(DMP- 1), dentin sialoprotein(DSP), ALP, osteocalcin(OC) mRNA expression reached peak on the 14 days in group A, and in group B on the 21 days. Bone sialoprotein(BSP) mRNA expression in both group A and B reached peak on the 21 days. BG45S5- SF membrane is able to support the proliferation and showed the potential of odontoblastic differentiation for hDPSC. This finding suggests that BG45S5-SF membrane was a kind of tissue engineering film material with the regeneration potential for pulp-dentine complex.

Evaluation of two human dental pulp stem cell cryopreservation methods.

Dental pulp is a promising source of mesenchymal stem cells for use in cell therapy and regenerative medicine. Methods for storing stem cells with minimum compromise of cell viability, differentiation capacity and function should be developed for clinical and research applications. The aim of this study was to evaluate whether human dental pulp stem cells (hDPSCs) isolated and cryopreserved for 1, 7 and 30 days maintain viability and expression of specific stem cell markers. Human dental pulp stem cells were isolated from 23 healthy patients aged 18 to 31 years. Dental pulp was enzymatically dissociated, and CD105+ cells were separated using the Miltenyi™ system. The hDPSCs were cryopreserved using the Kamath and Papaccio methods. Post-cryopreservation viability was measured by flow cytometry (7AAD) and by the expression of the phenotype markers CD105+/ CD73+, CD34-/CD45-. The Papaccio method showed greater cell viability for cells that had been frozen for 30 days (59.5%) than the Kamath method (56.2%), while the Kamath method provided better results for 1 day (65.5%) and 7 days (56%). Post-cryopreservation expression of the markers CD105+/CD34- was greater after 1 and 7 days with the Kamath method and CD105+/CD45- were expressed after all 3 cryopreservation times. There was greater expression of CD73+ in the hDPSCs after 1 and 7 days with the Kamath method, and after 30 days with the Papaccio method. These results suggest that hDPSCs express mesenchymal stem cell markers after cryopreservation. However, cryopreservation time may affect marker expression, probably by altering the spatialconfiguration of cell membrane proteins or by compromising cells at a certain level of differentiation.

Neural crest derived niche of human dental pulp stem cells promotes peripheral nerve regeneration and remyelination in animal model of critical sized sciatic nerve injury

Peripheral nerve injuries are a commonly encountered clinical problem and often result in long-term functional defects. The use of stem cells, easily accessible, capable of rapid expansion in culture as well as fully integrate into the host tissue and capable to differentiate in myelinating cells of the peripheral nervous system, represent an attractive therapeutic approach for the treatment of nerve injuries. Farther, stem cells sources sharing the same embryological origin of Schwann cells, might be considered a suitable tool. The aim of this study was to demonstrate the ability of a neuroectodermal sub-population of STRO-1+/c-Kit+/CD34+ hDPSCs (1, 2), most of which being positive for neural crest (P75NTR) and neural progenitor cells (nestin) markers, to differentiate into Schwann cells-like cells in vitro and to promote axonal regeneration in vivo. As a matter of fact, following culture in appropriate induction medium, STRO-1+/c-Kit+/CD34+ hDPSCs were able to commit towards Schwann cells express- ing P75NTR, GFAP and S100b. After transplantation in animal model of sciatic nerve defect, hDPSCs promoted axonal regeneration from proximal to distal stumps, providing guidance to newly formed myelinated nerve fibers, which led to functional recovery as measured by sustained gait improvement. Particularly, transplanted hDP- SCs engrafted into critical sized sciatic nerve defect, as revealed by the positive stain- ing against human nuclei, showed the expression of typical Schwann cells markers, S100b and GFAP. In conclusion this study demonstrates that STRO-1+/c-Kit+/CD34+ hDPSCs, associated to neural crest derivation, represent a promising source of stem cells for the treatment of demyelinating disorders and might provide a valid alternative tool for future clinical applications to achieve functional recovery after injury or peripheral neuropathies besides minimizing ethical issues.

Human dental pulp stem cells as a potential feeder layer for human embryonic stem cell culture

Background: Human embryonic stem (hES) cells are pluripotent, and can differentiate into three germ layers. Traditionally, cultures of hES cells are maintained in a system containing mouse embryonic fibroblasts as a feeder layer for support of undifferentiated growth. However, contamination by animal cells limits the use of hES cells. Objective: We evaluated the use of human dental pulp stem cells (hDPSCs) as a feeder layer for hES cell culture. It should be possible to obtain a new source of human mesenchymal stem cells for feeder cells to maintain undifferentiated growth of hES cells. Methods: hDPSCs from removed impacted wisdom teeth (third molars) were extracted, cultured, and characterized for mesenchymal stem cell properties. Furthermore, hDPSCs were used as a feeder layer for culturing Chula2 and Chula5 hES cell lines. Finally, hES cell lines grown on hDPSCs feeders were examined embryonic stem cell properties. Results: We found that hDPSCs, which have mesenchymal properties, can support undifferentiated growth of hES cell lines. After prolonged culture (passage 17), these hES cell lines still maintain ES cell properties including typical morphology seen in hES cells, the expression of pluripotency markers (Oct4, Sox2, Nanog, Rex1, SSEA-3, SSEA-4, TRA-1-60, and TRA-1-81), embryoid body formation and retention of a normal karyotype. Conclusion: hDPSCs, derived from the pulp tissue of impacted third molars, are a potential source of human feeder cells for the culture of undifferentiated hES cells.

Cytotoxic effects of various mineral trioxide aggregate formulations, calcium-enriched mixture and a new cement on human pulp stem cells.

This in vitro study compared the cytotoxic effects of three commercially available MTA formulations naming ProRoot MTA (PMTA), Angelus MTA (AMTA), and Root MTA (RMTA), with calcium-enriched mixture (CEM) cement and a new nanohybrid MTA (NMTA) on human dental pulp stem cells (DPSC). Four disc-shaped specimens of each material were prepared. After completion of setting, 2 different (neat and 1/2) elutes of the test materials were made. Then in each cavity of a 96-well plate, 3000 cells were seeded and incubated in a humidified incubator with 5% CO2 and 95% air at 37(°)C for 24 h. After this period, the culture medium of each well was replaced with 200 μL of test material elutes. Plain culture medium was used as the negative control and distilled water as the positive control group. Cell viability was assessed using 2, 5-diphenyl-SH-tetrazelium bromide colorimetric assay, aka Mosmann's tetrazolium toxicity (MTT) assay, at three time intervals (24, 48, and 72 h after mixing). Data were analyzed using the ANOVA and Tukey's post hoc test (P=0.05). After 24 h, the viability of cells in neat concentration had no significant differences (P>0.05) except for the NMTA. However, CEM and AMTA, at 1/2 concentration exerted significant proliferative effects on cells. At 48 and 72-h intervals, significant proliferation of DPSCs was seen in all samples, except for the NMTA which exerted toxic effects on cells. All of the three commercial MTAs and CEM cement showed comparative biocompatibility. However, NMTA had cytotoxic effects on DPSCs at all the time intervals.

Effects of equiaxial strain on the differentiation of dental pulp stem cells without using biochemical reagents.

During orthodontic treatments, applied mechanical forces create strain and result in tooth movement through the alveolar bone. This response to mechanical strain is a fundamental biological reaction. The present study evaluated the effect of equiaxial strain within the range of orthodontic forces on the osteogenic differentiation of human dental pulp stem cells (hDPSCs). Following isolation and culture of hDPSCs, 3rd passage cells were transferred on a silicone membrane covered with collagen. Cell adhesion to the membrane was evaluated under scanning electron microscope (SEM). Cells were divided into three groups: the first group was placed in a conventional culture medium, transferred to an equiaxial stretching device (3% strain for 2 weeks). The positive control was placed in an osteogenic medium with no mechanical strain. The negative control group was placed in the conventional culture medium with no mechanical strain either. Study groups were evaluated for expression ofosteogenic markers (Alkaline phosphatase and Osteopontin) with immunofluorescence and real time PCR. SEM images revealed optimal adhesion of cells to the silicone membrane. Immunofluorescence study demonstrated that osteocalcin expression occurred after 2 weeks in the two groups under mechanical and chemical signals. After application of equiaxial strain, level of expression of osteogenic markers was significantly higher than in the negative and positive control groups. Based on the study results, static equiaxial strain which mimics the types of orthodontic forces can result in differentiation of hDPSCs to osteoblasts. The results obtained may be used in cell therapy and tissue engineering.

In vitro cardiomyogenic differentiation of human dental pulp stem cells. The role of 5-azacytidine

Cellular cardiomyoplasty has been introduced as a potential therapy for treating heart failure and has generated significant interest in identifying various cell types capable of restoring the injured myocardium. Autologous human dental pulp stem cells (hDPSCs) show the ability to differentiate into various cell type such as osteoblasts1, chondroblasts, and adipoblasts. Aim of this study is to investigate the aptitude of hDPSCs to differentiate towards a cardiomyogenic phenotype in vitro. In this study we developed an efficient protocol for the generation of functional cardiomyocytes. Second passaged cells were treated with 5μM of 5-azacytidine2, a well-known demethylating agent, for 48h. Initial exposition of cultured hDPSCs to 5-azacytidine leads to development of embryoid bodies (EBs) expressing specific transcripts markers of the three germ layers as : ectoderm (MAP2), mesoderm (MSX1), and endoderm (PAX6) and various precardiac markers including MYH6, MESP. EBs subsequently plated onto gelatin-coated tissue culture dishes express cardiac markers as Nkx2.5 and connexin 43 determinated by PCR and immunofluorescence analysis at confocal microscopy, indicating that EBs can differentiate into functional cardiomyocytes. These results demonstrate that hDPSCs can be an easily source of stem cells able to undergo versus the cardiomyiogenic lineage.

Effect of platelet-rich plasma on cell proliferation and osteogenic activity of pulp stem cells

To evaluate the effects of different concentrations of platelet-rich plasma (PRP) on human dental pulp stem cells (hDPSC) proliferation and osteogenic differentiation activity so as to provide basis for future application of dental pulp stem cells and PRP in tissue engineering and bone repair therapy. hDPSC were isolated and cultivated in vitro. Flow cytometric analysis was carried out to test the expression of STRO-1.hDPSC were cultured in various concentrations of PRP (1%, 5%, 10%, 20%). At the 2nd and 6th day 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) was tested. Osteogenic differentiation of hDPSC was assessed using alkaline phosphatase (ALP) staining and Alizarin Red staining at the 7th and 14th day. Flow cytometric analysis demonstrated that 14.82% of hDPSC were STRO-1 positive. One percent to 20% PRP showed significant effect of promoting hDPSC proliferation. One percent to 10% PRP showed significant effect of promoting hDPSC osteogenic differentiation. Certain concentrations of PRP can promote hDPSC proliferate and osteogenic differentiate, and this finding suggests future application of dental pulp stem cells and PRP in bone tissue engineering.

A Cell-permeable Fusion Protein for the Mineralization of Human Dental Pulp Stem Cells

Human dental pulp stem cells (hDPSCs) are the only mesenchymal stem cells in pulp tissue that can differentiate into osteoblasts, odontoblasts, and adipose cells. The transcriptional co-activator with PDZ-binding motif (TAZ) protein has been reported to modulate osteogenic differentiation in mouse MSCs. Therefore, we examined whether the TAZ protein plays the same role in human pulp stem cells. In this study, TAZ was applied to cells directly with low-molecular-weight protamine (LMWP) as a cell-penetrating peptide (CPP). The LMWP-TAZ fusion proteins were expressed in an E. coli system with a pET-21b vector and efficiently transferred into hDPSCs without producing toxicity in the cells. The efficient uptake of TAZ was shown by Western blot with an anti-TAZ antibody, fluorescence-activated cell sorting, and confocal microscopy in live cells. The delivered TAZ protein increased osteogenic differentiation, as confirmed by alkaline phosphatase (ALP) staining, RT-PCR, and Western blotting. In addition, TAZ also inhibited adipogenic differentiation, regulating peroxisome proliferator-activated receptor-γ (PPAR-γ), lipoprotein lipase (LPL), and adipocyte fatty acid-binding protein (aP2) mRNA levels. These in vitro studies suggest that cell-permeable TAZ may be used as a specific regulator of hard-tissue differentiation.