Enhancing Dental Pulp Stem Cell Proliferation and Odontogenic Differentiation with Protein Phosphatase 1-Disrupting Peptide: An In Vitro Study.

The reparative and regenerative capabilities of dental pulp stem cells (DPSCs) are crucial for responding to pulp injuries, with protein phosphatase 1 (PP1) playing a significant role in regulating cellular functions pertinent to tissue healing. Accordingly, this study aimed to explore the effects of a novel cell-penetrating peptide Modified Sperm Stop 1-MSS1, that disrupts PP1, on the proliferation and odontogenic differentiation of DPSCs. Employing MSS1 as a bioportide, DPSCs were cultured and characterized for metabolic activity, cell proliferation, and cell morphology alongside the odontogenic differentiation through gene expression and alkaline phosphatase (ALP) activity analysis. MSS1 exposure induced early DPSC proliferation, upregulated genes related to odontogenic differentiation, and increased ALP activity. Markers associated with early differentiation events were induced at early culture time points and those associated with matrix mineralization were upregulated at mid-culture stages. This investigation is the first to document the potential of a PP1-disrupting bioportide in modulating DPSC functionality, suggesting a promising avenue for enhancing dental tissue regeneration and repair.

Novel L-(CaP-ZnP)/SA Nanocomposite Hydrogel with Dual Anti-Inflammatory and Mineralization Effects for Efficient Vital Pulp Therapy.

Background: Vital pulp therapy (VPT) is considered a conservative treatment for preserving pulp viability in caries and trauma-induced pulpitis. However, Mineral trioxide aggregate (MTA) as the most frequently used repair material, exhibits limited efficacy under inflammatory conditions. This study introduces an innovative nanocomposite hydrogel, tailored to simultaneously target anti-inflammation and dentin mineralization, aiming to efficiently preserve vital pulp tissue. Methods: The L-(CaP-ZnP)/SA nanocomposite hydrogel was designed by combining L-Arginine modified calcium phosphate/zinc phosphate nanoparticles (L-(CaP-ZnP) NPs) with sodium alginate (SA), and was characterized with TEM, SEM, FTIR, EDX, ICP-AES, and Zeta potential. In vitro, we evaluated the cytotoxicity and anti-inflammatory properties. Human dental pulp stem cells (hDPSCs) were cultured with lipopolysaccharide (LPS) to induce an inflammatory response, and the cell odontogenic differentiation was measured and possible signaling pathways were explored by alkaline phosphatase (ALP)/alizarin red S (ARS) staining, qRT-PCR, immunofluorescence staining, and Western blotting, respectively. In vivo, a pulpitis model was utilized to explore the potential of the L-(CaP-ZnP)/SA nanocomposite hydrogel in controlling pulp inflammation and enhancing dentin mineralization by Hematoxylin and eosin (HE) staining and immunohistochemistry staining. Results: In vitro experiments revealed that the nanocomposite hydrogel was synthesized successfully and presented desirable biocompatibility. Under inflammatory conditions, compared to MTA, the L-(CaP-ZnP)/SA nanocomposite hydrogel demonstrated superior anti-inflammatory and pro-odontogenesis effects. Furthermore, the nanocomposite hydrogel significantly augmented p38 phosphorylation, implicating the involvement of the p38 signaling pathway in pulp repair. Significantly, in a rat pulpitis model, the L-(CaP-ZnP)/SA nanocomposite hydrogel downregulated inflammatory markers while upregulating mineralization-related markers, thereby stimulating the formation of robust reparative dentin. Conclusion: The L-(CaP-ZnP)/SA nanocomposite hydrogel with good biocompatibility efficiently promoted inflammation resolution and enhanced dentin mineralization by activating p38 signal pathway, as a pulp-capping material, offering a promising and advanced solution for treatment of pulpitis.

Apoptotic Extracellular Vesicles from Supernumerary Tooth-Derived Pulp Stem Cells Transfer COL1A1 to Promote Angiogenesis via PI3K/Akt/VEGF Pathway.

Purpose: Angiogenesis is a tightly controlled process that initiates the formation of new vessels and its dysfunction can lead to life-threatening diseases. Apoptotic extracellular vesicles (ApoEVs) have emerged as a proangiogenic agent with high safety and isolation efficiency profile, and ApoEVs from supernumerary tooth-derived pulp stem cells (SNTSC-ApoEVs) have their unique advantages with an easily accessible parental cell source and non-invasive cell harvesting. However, the detailed characteristics of SNTSC-ApoEVs are largely unknown. This study aimed to investigate the proangiogenic capacity and function molecule of SNTSC-ApoEVs. Methods: SNTSC-ApoEVs were isolated and characterized. In vitro effects of SNTSC-ApoEVs on the proliferation, migration, and tube formation of human umbilical vein endothelial cells (HUVECs) were evaluated by CCK-8, wound healing, transwell, and tube formation assays. The mRNA and protein levels of proangiogenic genes were quantified by qRT-PCR, Western blot, and immunofluorescence analysis. A Matrigel plug model was established in 6-week-old male nu/nu mice for one week, and the in vivo impact of SNTSC-ApoEVs on micro-vessel formation was assessed by histological analysis. Proteomic analysis and RNA sequencing were performed to explore the active ingredients and underlying mechanisms. Results: SNTSC-ApoEVs enhanced the proliferation, migration, and angiogenesis of HUVECs in vitro. In the Matrigel plug model in vivo, SNTSC-ApoEVs promoted CD31-positive luminal structure formation. Apart from expressing general ApoEV markers, SNTSC-ApoEVs were enriched with multiple proteins related to extracellular matrix-cell interactions. Mechanistically, SNTSC-ApoEVs transferred COL1A1 to HUVECs and promoted endothelial functions by activating the PI3K/Akt/VEGF cascade. Conclusion: SNTSC-ApoEVs can promote angiogenesis by transferring the functional molecule COL1A1 and activating the PI3K/Akt/VEGF pathway, making SNTSC-ApoEVs a promising strategy for the treatment of angiogenesis-related diseases.

A new subtype of artificial cell-derived vesicles from dental pulp stem cells with the bioequivalence and higher acquisition efficiency compared to extracellular vesicles.

Extracellular vesicles (EVs) derived from dental pulp stem cells (DPSC) have been shown an excellent efficacy in a variety of disease models. However, current production methods fail to meet the needs of clinical treatment. In this study, we present an innovative approach to substantially enhance the production of 'Artificial Cell-Derived Vesicles (ACDVs)' by extracting and purifying the contents released by the DPSC lysate, namely intracellular vesicles. Comparative analysis was performed between ACDVs and those obtained through ultracentrifugation. The ACDVs extracted from the cell lysate meet the general standard of EVs and have similar protein secretion profile. The new ACDVs also significantly promoted wound healing, increased or decreased collagen regeneration, and reduced the production of inflammatory factors as the EVs. More importantly, the extraction efficiency is improved by 16 times compared with the EVs extracted using ultracentrifuge method. With its impressive attributes, this new subtype of ACDVs emerge as a prospective candidate for the future clinical applications in regenerative medicine.

Regenerative Potential of Dental Pulp Stem Cells in Response to a Bioceramic Dental Sealer and Photobiomodulation: An In Vitro Study.

AIMS: This study aims to assess the synergistic effect of utilizing a bioceramic sealer, NeoPutty, with photobiomodulation (PBM) on dental pulp stem cells (DPSCs) for odontogenesis. MATERIALS AND METHODS: Dental pulp stem cells were collected from 10 premolars extracted from healthy individuals. Dental pulp stem cells were characterized using an inverted-phase microscope to detect cell shape and flow cytometry to detect stem cell-specific surface antigens. Three experimental groups were examined: the NP group, the PBM group, and the combined NP and PBM group. A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) experiment was conducted to assess the viability of DPSCs. The odontogenic differentiation potential was analyzed using Alizarin red staining, RT-qPCR analysis of odontogenic genes DMP-1, DSPP, and alkaline phosphatase (ALP), and western blot analysis for detecting BMP-2 and RUNX-2 protein expression. An analysis of variance (ANOVA) followed by a post hoc t-test was employed to examine and compare the mean values of the results. RESULTS: The study showed a notable rise in cell viability when NP and PBM were used together. Odontogenic gene expression and the protein expression of BMP-2 and RUNX-2 were notably increased in the combined group. The combined effect of NeoPutty and PBM was significant in enhancing the odontogenic differentiation capability of DPSCs. CONCLUSION: The synergistic effect of NeoPutty and PBM produced the most positive effect on the cytocompatibility and odontogenic differentiation potential of DPSCs. CLINICAL SIGNIFICANCE: Creating innovative regenerative treatments to efficiently and durably repair injured dental tissues. How to cite this article: Alshawkani HA, Mansy M, Al Ankily M, et al. Regenerative Potential of Dental Pulp Stem Cells in Response to a Bioceramic Dental Sealer and Photobiomodulation: An In Vitro Study. J Contemp Dent Pract 2024;25(4):313-319.

Enhancement of the chondrogenic differentiation capacity of human dental pulp stem cells via chondroitin sulfate-coated polycaprolactone-MWCNT nanofibers.

Most of the conditions involving cartilaginous tissues are irreversible and involve degenerative processes. The aim of the present study was to fabricate a biocompatible fibrous and film scaffolds using electrospinning and casting techniques to induce chondrogenic differentiation for possible application in cartilaginous tissue regeneration. Polycaprolactone (PCL) electrospun nanofibrous scaffolds and PCL film were fabricated and incorporated with multi-walled carbon nanotubes (MWCNTs). Thereafter, coating of chondroitin sulfate (CS) on the fibrous and film structures was applied to promote chondrogenic differentiation of human dental pulp stem cells (hDPSCs). First, the morphology, hydrophilicity and mechanical properties of the scaffolds were characterized by scanning electron microscopy (SEM), spectroscopic characterization, water contact angle measurements and tensile strength testing. Subsequently, the effects of the fabricated scaffolds on stimulating the proliferation of human dental pulp stem cells (hDPSCs) and inducing their chondrogenic differentiation were evaluated via electron microscopy, flow cytometry and RT‒PCR. The results of the study demonstrated that the different forms of the fabricated PCL-MWCNTs scaffolds analyzed demonstrated biocompatibility. The nanofilm structures demonstrated a higher rate of cellular proliferation, while the nanofibrous architecture of the scaffolds supported the cellular attachment and differentiation capacity of hDPSCs and was further enhanced with CS addition. In conclusion, the results of the present investigation highlighted the significance of this combination of parameters on the viability, proliferation and chondrogenic differentiation capacity of hDPSCs seeded on PCL-MWCNT scaffolds. This approach may be applied when designing PCL-based scaffolds for future cell-based therapeutic approaches developed for chondrogenic diseases.

Research and development of microenvironment's influence on stem cells from the apical papilla - construction of novel research microdevices: tooth-on-a-chip.

Stem cells are crucial in tissue engineering, and their microenvironment greatly influences their behavior. Among the various dental stem cell types, stem cells from the apical papilla (SCAPs) have shown great potential for regenerating the pulp-dentin complex. Microenvironmental cues that affect SCAPs include physical and biochemical factors. To research optimal pulp-dentin complex regeneration, researchers have developed several models of controlled biomimetic microenvironments, ranging from in vivo animal models to in vitro models, including two-dimensional cultures and three-dimensional devices. Among these models, the most powerful tool is a microfluidic microdevice, a tooth-on-a-chip with high spatial resolution of microstructures and precise microenvironment control. In this review, we start with the SCAP microenvironment in the regeneration of pulp-dentin complexes and discuss research models and studies related to the biological process.

Superoxide dismutase 1-modified dental pulp stem cells alleviate high-altitude pulmonary edema by inhibiting oxidative stress through the Nrf2/HO-1 pathway.

High-altitude pulmonary edema (HAPE) is a deadly form of altitude sickness, and there is no effective treatment for HAPE. Dental pulp stem cells (DPSCs) are a type of mesenchymal stem cell isolated from dental pulp tissues and possess various functions, such as anti-inflammatory and anti-oxidative stress. DPSCs have been used to treat a variety of diseases, but there are no studies on treating HAPE. In this study, Sprague-Dawley rats were exposed to acute low-pressure hypoxia to establish the HAPE model, and SOD1-modified DPSCs (DPSCsHiSOD1) were administered through the tail vein. Pulmonary arterial pressure, lung water content (LWC), total lung protein content of bronchoalveolar lavage fluid (BALF) and lung homogenates, oxidative stress, and inflammatory indicators were detected to evaluate the effects of DPSCsHiSOD1 on HAPE. Rat type II alveolar epithelial cells (RLE-6TN) were used to investigate the effects and mechanism of DPSCsHiSOD1 on hypoxia injury. We found that DPSCs could treat HAPE, and the effect was better than that of dexamethasone treatment. SOD1 modification could enhance the function of DPSCs in improving the structure of lung tissue, decreasing pulmonary arterial pressure and LWC, and reducing the total lung protein content of BALF and lung homogenates, through anti-oxidative stress and anti-inflammatory effects. Furthermore, we found that DPSCsHiSOD1 could protect RLE-6TN from hypoxic injury by reducing the accumulation of reactive oxygen species (ROS) and activating the Nrf2/HO-1 pathway. Our findings confirm that SOD1 modification could enhance the anti-oxidative stress ability of DPSCs through the Nrf2/HO-1 signalling pathway. DPSCs, especially DPSCsHiSOD1, could be a potential treatment for HAPE. Schematic diagram of the antioxidant stress mechanism of DPSCs in the treatment of high-altitude pulmonary edema. DPSCs can alleviate oxidative stress by releasing superoxide dismutase 1, thereby reducing ROS production and activating the Nrf2/HO-1 signalling pathway to ameliorate lung cell injury in HAPE.

An innovative cell-based transplantation therapy for an immature permanent tooth in an adult: a case report.

BACKGROUND: Immature teeth with necrotic pulps present multiple challenges to clinicians. In such cases, regenerative endodontic procedures (REPs) may be a favorable strategy. Cells, biomaterial scaffolds, and signaling molecules are three key elements of REPs. Autologous human dental pulp cells (hDPCs) play an important role in pulp regeneration. In addition, autologous platelet concentrates (APCs) have recently been demonstrated as effective biomaterial scaffolds in regenerative dentistry, whereas the latest generation of APCs-concentrated growth factor (CGF), especially liquid phase CGF (LPCGF)-has rarely been reported in REPs. CASE PRESENTATION: A 31-year-old woman presented to our clinic with the chief complaint of occlusion discomfort in the left mandibular posterior region for the past 5 years. Tooth #35 showed no pulp vitality and had a periodontal lesion, and radiographic examination revealed that the tooth exhibited extensive periapical radiolucency with an immature apex and thin dentin walls. REP was implemented via transplantation of autologous hDPCs with the aid of LPCGF. The periodontal lesion was managed with simultaneous periodontal surgery. After the treatment, the tooth was free of any clinical symptoms and showed positive results in thermal and electric pulp tests at 6- and 12-month follow-ups. At 12-month follow-up, radiographic evidence and three-dimensional models, which were reconstructed using Mimics software based on cone-beam computed tomography, synergistically confirmed bone augmentation and continued root development, indicating complete disappearance of the periapical radiolucency, slight lengthening of the root, evident thickening of the canal walls, and closure of the apex. CONCLUSION: hDPCs combined with LPCGF represents an innovative and effective strategy for cell-based regenerative endodontics.

Human dental pulp stem cells differentiation into odontoblast and osteoblast-like cells on scaffolds contain bioactive materials.

Epigenetic change has been found to play an important role in cell differentiation and regulation and the dental pulp stem cell in tissue engineering is gaining attention due to the ability of cells to differentiate into odontoblast and other cells. This study evaluated the influence of poly L- lactic acid with hydroxyapatite-coated with polyaniline scaffold (PLLA/HA/PANI) on dental pulp stem cell (DPSC) proliferation and differentiation. After scaffold preparation and DPSCs seeding, the cells proliferation and differentiation were evaluated by immunocytochemistry assay and cell viability was measured by cytotoxicity / MTT assay. The results showed (PLLA/HA/PANI) scaffold facilitates DPSC proliferation and differentiation with gene expression. This finding underscores the promise of this biomaterial combination as a scaffold for dental tissue regeneration and application.

Comparative cell viability of dentin-bonding adhesive systems on human dental pulp stem cells: time-dependent analysis.

BACKGROUND: Restorative materials are in prolonged contact with living tissues such as oral mucosa, dentin, pulp, periodontal, and periapical tissues. Therefore, the potentially harmful effects of these materials and their components on oral tissues should be evaluated before clinical use. This study aimed to compare the cell viability of different adhesive systems (ASs) on human dental pulp stem cells (hDPSCs). METHODS: Three ASs that combining methacryloyloxydecyl dihydrogen phosphate (MDP) monomer with new hydrophilic amide monomers [Clearfil Universal Bond Quick(CUBQ), Kuraray Noritake], self-reinforcing 3D monomer [Bond Force II(BFII), Tokuyama)], and dual-cure property [Futurabond DC(FBDC), VOCO] were used. Three (n = 3) samples were prepared for each group. Dental pulp stem cells were isolated from ten patients' extracted third molar teeth. Samples were incubated in Dulbecco's modified Eagle's medium (DMEM) for 24 h (h), 72 h, and 7 days (d) to obtain extracts. For the control group, cells were cultured without DBA samples. Cell viability of ASs extracts was measured using a cell proliferation detection kit (WST-1, Roche). Statistical analysis was performed using two-way ANOVA and post-hoc (Duncan) tests (p < 0.05). RESULTS: At 24 and 72 h statistically significant differences were determined between control and BFII, control and FBDC groups (p < 0.05), while no differences between control and CUBQ groups (p > 0.05). On the 7th d, statistically significant differences were found between the control and experimental groups (p < 0.05), while no differences between experimental groups (p > 0.05). A statistically significant difference was detected for the BFII group over the three-time interval (p < 0.05). The lowest cell viability was observed for the FBDC group at 24 h, and the difference was statistically significant when compared with 72 h and 7th d (p < 0.05). CONCLUSION: All ASs showed different cell viability values at various exposure times. It should be taken into consideration that pH values, as well as the contents of ASs, have a significant effect on the cell viability.

Heparin-Functionalized Bioactive Glass to Harvest Endogenous Growth Factors for Pulp Regeneration.

Pulp and periapical diseases can lead to the cessation of tooth development, resulting in compromised tooth structure and functions. Despite numerous efforts to induce pulp regeneration, effective strategies are still lacking. Growth factors (GFs) hold considerable promise in pulp regeneration due to their diverse cellular regulatory properties. However, the limited half-lives and susceptibility to degradation of exogenous GFs necessitate the administration of supra-physiological doses, leading to undesirable side effects. In this research, a heparin-functionalized bioactive glass (CaO-P2O5-SiO2-Heparin, abbreviated as PSC-Heparin) with strong bioactivity and a stable neutral pH is developed as a promising candidate to addressing challenges in pulp regeneration. Fourier transform infrared spectroscopy, X-ray photoelectron spectroscopy, and thermogravimetric analysis reveal the successful synthesis of PSC-Heparin. Scanning electron microscopy and X-ray diffraction show the hydroxyapatite formation can be observed on the surface of PSC-Heparin after soaking in simulated body fluid for 12 h. PSC-Heparin is capable of harvesting various endogenous GFs and sustainably releasing them over an extended duration by the enzyme-linked immunosorbent assay. Cytological experiments show that developed PSC-Heparin can facilitate the adhesion, migration, proliferation, and odontogenic differentiation of stem cells from apical papillae. Notably, the histological analysis of subcutaneous implantation in nude mice demonstrates PSC-Heparin is capable of promoting the odontoblast-like layers and pulp-dentin complex formation without the addition of exogenous GFs, which is vital for clinical applications. This work highlights an effective strategy of harvesting endogenous GFs and avoiding the involvement of exogenous GFs to achieve pulp-dentin complex regeneration, which may open a new horizon for regenerative endodontic therapy.

Cytotoxic effects on human dental pulp stem Cells after exposure to adhesive bonding agents.

Studies regarding cytotoxic effects attributed to the use of adhesive bonding agents on pulp tissue are not conclusive. To point out whether these materials are safe for clinical use, in vivo exposure of dental pulp to adhesive bonding agents was simulated using an experimental setup in which Human Dental Pulp Stem Cells (hDPSC) are exposed to the action of two kinds of adhesives: self-etching adhesives and two-step bonding agents through a dentine barrier. Cytotoxic effects on these cells were evaluated by MTT assay protocol and fluorescence microscopy, and their results were contrasted to those obtained through Raman spectra taken on single hDPSCs. Overall, no significant cytotoxic effects were observed by combining all the techniques, and cell viability close to 90% was achieved for a dentine barrier of at least 1 mm thick. Moreover, Raman spectroscopy was able to detect structural DNA damage in some dental pulp cells when exposed to two-step bonding agents, suggesting that this technique could be considered a complementary tool with the potential to evaluate cell toxicity beyond cell viability.

Overexpression of programmed cell death ligand 1 reduces LPS-induced inflammatory cytokine upregulation and enhances osteo/odontogenic-differentiation of human dental pulp stem cells via upregulation of CCCTC-binding factor.

OBJECTIVE: The aim of this study was to explore the effect and mechanism of programmed cell death ligand 1 (PD-L1) in promoting the proliferation and osteo/odontogenic-differentiation of human dental pulp stem cells (hDPSCs) by mediating CCCTC-binding factor (CTCF) expression. DESIGN: The interaction between PD-L1 and CTCF was verified through co-immunoprecipitation. hDPSCs transfected with PD-L1 overexpression and CTCF knockdown vectors were treated with lipopolysaccharide or an osteogenic-inducing medium. Inflammatory cytokines and osteo/odontogenic-differentiation related genes were measured. Osteo/odontogenic-differentiation of hDPSCs was assessed using alkaline phosphatase (ALP) and alizarin red S staining. RESULTS: Overexpression of PD-L1 inhibited LPS-induced pro-inflammatory cytokine upregulation, cell proliferation, ALP activity, and calcium deposition in hDPSCs and elevated the expression of osteo/odontogenic-differentiation related genes; however, such expression patterns could be reversed by CTCF knockdown. Co-immunoprecipitation results confirmed the binding of PD-L1 to CTCF, indicating that PD-L1 overexpression in hDPSCs increases CTCF expression, thus inhibiting the inflammatory response and increasing osteo/odontogenic-differentiation of hDPSCs. CONCLUSION: PD-L1 overexpression in hDPSCs enhances the proliferation and osteo/odontogenic-differentiation of hDPSCs and inhibit the inflammatory response by upregulating CTCF expression.

Green synthesis of nanohydroxyapatite with Elaeagnus angustifolia L. extract as a metronidazole nanocarrier for in vitro pulpitis model treatment.

The aim of this study is to introduce a dental capping agent for the treatment of pulp inflammation (pulpitis). Nanohydroxyapatite with Elaeagnus angustifolia L. extract (nHAEA) loaded with metronidazole (nHAEA@MTZ) was synthesized and evaluated using a lipopolysaccharide (LPS) in vitro model of pulpitis. nHAEA was synthesized through sol-gel method and analyzed using Scanning Electron Microscopy, Transmission Electron Microscopy, and Brunauer Emmett Teller. Inflammation in human dental pulp stem cells (HDPSCs) induced by LPS. A scratch test assessed cell migration, RT PCR measured cytokines levels, and Alizarin red staining quantified odontogenesis. The nHAEA nanorods were 17-23 nm wide and 93-146 nm length, with an average pore diameter of 27/312 nm, and a surface area of 210.89 m2/g. MTZ loading content with controlled release, suggesting suitability for therapeutic applications. nHAEA@MTZ did not affect the odontogenic abilities of HDPSCs more than nHAEA. However, it was observed that nHAEA@MTZ demonstrated a more pronounced anti-inflammatory effect. HDPSCs treated with nanoparticles exhibited improved migration compared to other groups. These findings demonstrated that nHAEA@MTZ could be an effective material for pulp capping and may be more effective than nHAEA in reducing inflammation and activating HDPSCs to enhance pulp repair after pulp damage.

A Preliminary Investigation into the Effect of Low-Energy Lasers on Dental Pulp Stem Cell Proliferation and the Associated Mechanism.

BACKGROUND: Dental pulp stem cells (DPSCs) have self-renewal and multidirectional differentiation potentials. As such, DPSCs have a wide range of clinical applications. Low-level laser therapy (LLLT) has positive photobiostimulatory effects on cell proliferation, angiogenesis, osteogenic differentiation, bone regeneration, and fracture healing. However, there have been few studies on the effect of low-energy lasers on DPSC proliferation. METHODS: DPSCs were obtained from dental pulp tissue. The effects of LLLT on the proliferation of DPSCs and the associated mechanisms were investigated by in vitro culture and laser irradiation. RESULTS: LLLT with energy densities of 3.5 J/cm2 and 14 J/cm2promoted the proliferation of DPSCs. Differential protein expression studies suggested the stimulation of DPSC proliferation by LLLT involved the PI3K-Akt and Rap1 signaling pathways, as well as the apoptosis-related pathway. CONCLUSION: This preliminary study demonstrated that low-energy lasers have a pro-proliferative effect on DPSCs, and identified possible associated mechanisms. Our findings provide a theoretical basis for the clinical application of DPSCs and suggest novel strategies for the treatment of related diseases.

Scaffold Application for Bone Regeneration with Stem Cells in Dentistry: Literature Review.

Bone tissue injuries within oral and dental contexts often present considerable challenges because traditional treatments may not be able to fully restore lost or damaged bone tissue. Novel approaches involving stem cells and targeted 3D scaffolds have been investigated in the search for workable solutions. The use of scaffolds in stem cell-assisted bone regeneration is a crucial component of tissue engineering techniques designed to overcome the drawbacks of traditional bone grafts. This study provides a detailed review of scaffold applications for bone regeneration with stem cells in dentistry. This review focuses on scaffolds and stem cells while covering a broad range of studies explaining bone regeneration in dentistry through the presentation of studies conducted in this field. The role of different stem cells in regenerative medicine is covered in great detail in the reviewed literature. These studies have addressed a wide range of subjects, including the effects of platelet concentrates during dental surgery or specific combinations, such as human dental pulp stem cells with scaffolds for animal model bone regeneration, to promote bone regeneration in animal models. Noting developments, research works consider methods to improve vascularization and explore the use of 3D-printed scaffolds, secretome applications, mesenchymal stem cells, and biomaterials for oral bone tissue regeneration. This thorough assessment outlines possible developments within these crucial regenerative dentistry cycles and provides insights and suggestions for additional study. Furthermore, alternative creative methods for regenerating bone tissue include biophysical stimuli, mechanical stimulation, magnetic field therapy, laser therapy, nutritional supplements and diet, gene therapy, and biomimetic materials. These innovative approaches offer promising avenues for future research and development in the field of bone tissue regeneration in dentistry.

Dynamic Alterations in Acetylation and Modulation of Histone Deacetylase Expression Evident in the Dentine-Pulp Complex during Dentinogenesis.

Epigenetic modulation, including histone modification, alters gene expression and controls cell fate. Histone deacetylases (HDACs) are identified as important regulators of dental pulp cell (DPC) mineralisation processes. Currently, there is a paucity of information regarding the nature of histone modification and HDAC expression in the dentine-pulp complex during dentinogenesis. The aim of this study was to investigate post-translational histone modulation and HDAC expression during DPC mineralisation and the expression of Class I/II HDACs during tooth development and in adult teeth. HDAC expression (isoforms -1 to -6) was analysed in mineralising primary rat DPCs using qRT-PCR and Western blot with mass spectrometry being used to analyse post-translational histone modifications. Maxillary molar teeth from postnatal and adult rats were analysed using immunohistochemical (IHC) staining for HDACs (1-6). HDAC-1, -2, and -4 protein expression increased until days 7 and 11, but decreased at days 14 and 21, while other HDAC expression increased continuously for 21 days. The Class II mineralisation-associated HDAC-4 was strongly expressed in postnatal sample odontoblasts and DPCs, but weakly in adult teeth, while other Class II HDACs (-5, -6) were relatively strongly expressed in postnatal DPCs and adult odontoblasts. Among Class I HDACs, HDAC-1 showed high expression in postnatal teeth, notably in ameloblasts and odontoblasts. HDAC-2 and -3 had extremely low expression in the rat dentine-pulp complex. Significant increases in acetylation were noted during DPC mineralisation processes, while trimethylation H3K9 and H3K27 marks decreased, and the HDAC-inhibitor suberoylanilide hydroxamic acid (SAHA) enhanced H3K27me3. These results highlight a dynamic alteration in histone acetylation during mineralisation and indicate the relevance of Class II HDAC expression in tooth development and regenerative processes.

Platelet-rich fibrin (PRF) modified nano-hydroxyapatite/chitosan/gelatin/alginate scaffolds increase adhesion and viability of human dental pulp stem cells (DPSC) and osteoblasts derived from DPSC.

Bone tissue regeneration strategies have incorporated the use of natural polymers, such as hydroxyapatite (nHA), chitosan (CH), gelatin (GEL), or alginate (ALG). Additionally, platelet concentrates, such as platelet-rich fibrin (PRF) have been suggested to improve scaffold biocompatibility. This study aimed to develop scaffolds composed of nHA, GEL, and CH, with or without ALG and lyophilized PRF, to evaluate the scaffold's properties, growth factor release, and dental pulp stem cells (DPSC), and osteoblast (OB) derived from DPSC viability. Four scaffold variations were synthesized and lyophilized. Then, degradation, swelling profiles, and morphological analysis were performed. Furthermore, PDGF-BB and FGF-B growth factors release were quantified by ELISA, and cytotoxicity and cell viability were evaluated. The swelling and degradation profiles were similar in all scaffolds, with pore sizes ranging between 100 and 250 µm. FGF-B and PDGF-BB release was evidenced after 24 h of scaffold immersion in cell culture medium. DPSC and OB-DPSC viability was notably increased in PRF-supplemented scaffolds. The nHA-CH-GEL-PRF scaffold demonstrated optimal physical-biological characteristics for stimulating DPSC and OB-DPSC cell viability. These results suggest lyophilized PRF improves scaffold biocompatibility for bone tissue regeneration purposes.

Intracranial graft of bioresorbable polymer scaffolds loaded with human Dental Pulp Stem Cells in stab wound murine injury model.

The prevalence of central nervous system (CNS) dysfunction as a result of disease or trauma remains a clinically unsolved problem which is raising increased awareness in our aging society. Human Dental Pulp Stem Cells (hDPSCs) are excellent candidates to be used in tissue engineering and regenerative therapies of the CNS due to their neural differentiation ability and lack of tumorigenicity. Accordingly, they have been successfully used in animal models of spinal cord injury, stroke and peripheral neuropathies. The ideal therapy in brain injury should combine strategies aiming to protect the damaged lesion and, at the same time, accelerate brain tissue regeneration, thus promoting fast recovery while minimizing side or long-term effects. The use of bioresorbable nanopatterned poly(lactide-co-ɛ-caprolactone) (PLCL) polymeric scaffolds as hDPCSs carriers can represent an advantage for tissue regeneration. In this chapter, we describe the surgical procedures to implant functionalized bioresorbable scaffolds loaded with hDPSCs to improve the brain lesion microenvironment in an intracranial stab wound injury model severing the rostral migratory stream (RMS) that connects the brain subventricular zone (SVZ) and the olfactory bulb in nude mice. Additionally, we also describe the technical steps after animal sacrifice for histological tissue observation and characterization.