Dental Tissue Engineering Research Articles

Bioprinting of alginate-carboxymethyl chitosan scaffolds for enamel tissue engineering in vitro

Tissue engineering offers a great potential in regenerative dentistry and to this end, three dimensional (3D) bioprinting has been emerging nowadays to enable the incorporation of living cells into the biomaterials (such a mixture is referred as a bioink in the literature) to create scaffolds. However, the bioinks available for scaffold bioprinting are limited, particularly for dental tissue engineering, due to the complicated, yet compromised, printability, mechanical and biological properties simultaneously imposed on the bioinks. This paper presents our study on the development of a novel bioink from carboxymethyl chitosan (CMC) and alginate (Alg) for bioprinting scaffolds for enamel tissue regeneration. CMC was used due to its antibacterial ability and superior cell interaction properties, while Alg was added to enhance the printability and mechanical properties as well as to regulate the degradation rate. The bioinks with three mixture ratios of Alg and CMC (2–4, 3–3 and 4–2) were prepared, and then printed into the calcium chloride crosslinker solution (100 mM) to form a 3D structure of scaffolds. The printed scaffolds were characterized in terms of structural, swelling, degradation, and mechanical properties, followed by their in vitro characterization for enamel tissue regeneration. The results showed that the bioinks with higher concentrations of Alg were more viscous and needed higher pressure for printing; while the printed scaffolds were highly porous and showed a high degree of printability and structural integrity. The hydrogels with higher CMC ratios had higher swelling ratios, faster degradation rates, and lower compressive modulus. Dental epithelial cell line, HAT-7, could maintain high viability in the printed constructs after 1, 7 and 14 d of culture. HAT-7 cells were also able to maintain their morphology and secrete alkaline phosphatase after 14 d of culture in the 3D printed scaffolds, suggesting the capacity of these cells for mineral deposition and enamel-like tissue formation. Among all combinations Alg4%–CMC2% and in a less degree 2%Alg–4%CMC showed the higher potential to promote ameloblast differentiation, Ca and P deposition and matrix mineralization in vitro. Taken together, Alg-CMC has been illustrated to be suitable to print scaffolds with dental epithelial cells for enamel tissue regeneration.

Engineering Dental Tissues Using Biomaterials with Piezoelectric Effect: Current Progress and Future Perspectives

Dental caries and traumatic injuries to teeth may cause irreversible inflammation and eventual death of the dental pulp. Nevertheless, predictably, repair and regeneration of the dentin-pulp complex remain a formidable challenge. In recent years, smart multifunctional materials with antimicrobial, anti-inflammatory, and pro-regenerative properties have emerged as promising approaches to meet this critical clinical need. As a unique class of smart materials, piezoelectric materials have an unprecedented advantage over other stimuli-responsive materials due to their inherent capability to generate electric charges, which have been shown to facilitate both antimicrobial action and tissue regeneration. Nonetheless, studies on piezoelectric biomaterials in the repair and regeneration of the dentin-pulp complex remain limited. In this review, we summarize the biomedical applications of piezoelectric biomaterials in dental applications and elucidate the underlying molecular mechanisms contributing to the biological effect of piezoelectricity. Moreover, we highlight how this state-of-the-art can be further exploited in the future for dental tissue engineering.

Effects of Metformin Delivery via Biomaterials on Bone and Dental Tissue Engineering.

Bone tissue engineering is a promising approach that uses seed-cell-scaffold drug delivery systems to reconstruct bone defects caused by trauma, tumors, or other diseases (e.g., periodontitis). Metformin, a widely used medication for type II diabetes, has the ability to enhance osteogenesis and angiogenesis by promoting cell migration and differentiation. Metformin promotes osteogenic differentiation, mineralization, and bone defect regeneration via activation of the AMP-activated kinase (AMPK) signaling pathway. Bone tissue engineering depends highly on vascular networks for adequate oxygen and nutrition supply. Metformin also enhances vascular differentiation via the AMPK/mechanistic target of the rapamycin kinase (mTOR)/NLR family pyrin domain containing the 3 (NLRP3) inflammasome signaling axis. This is the first review article on the effects of metformin on stem cells and bone tissue engineering. In this paper, we review the cutting-edge research on the effects of metformin on bone tissue engineering. This includes metformin delivery via tissue engineering scaffolds, metformin-induced enhancement of various types of stem cells, and metformin-induced promotion of osteogenesis, angiogenesis, and its regulatory pathways. In addition, the dental, craniofacial, and orthopedic applications of metformin in bone repair and regeneration are also discussed.

Naringenin stimulates osteogenic/odontogenic differentiation and migration of human dental pulp stem cells

Background/purposeNaringenin, a naturally occurring flavanone in citrus fruits, regulates bone formation by bone marrow-derived mesenchymal stem cells. The purpose of this study was to characterize the effects of naringenin on some biological behaviors of human dental pulp stem cells (HDPSCs). Materials and methodsHDPSCs were cultured in osteogenic differentiation medium and osteo/odontogenic differentiation and mineralization were analyzed by alkaline phosphatase (ALP) staining and Alizarin Red S (ARS) staining. The migration of HDPSCs was evaluated by transwell chemotactic migration assays and scratch wound healing migration assay. Using tooth slice/scaffold model, we assessed the in vivo odontogenic differentiation potential of HDPSCs. ResultsWe have demonstrated that naringenin increases the osteogenic/odontogenic differentiation of HDPSCs through regulation of osteogenic-related proteins and the migratory ability of HDPSCs through stromal cell derived factor-1 (SDF-1)/C-X-C chemokine receptor type 4 (CXCR4) axis. Moreover, naringenin promotes the expression of dentin matrix acidic phosphoprotein-1 (DMP-1) and dentin sialophosphoprotein (DSPP) in HDPSCs seeded on tooth slice/scaffolds that are subcutaneously implanted into immunodeficient mice. ConclusionOur present study suggests that naringenin promotes migration and osteogenic/odontogenic differentiation of HDPSCs and may serve as a promising candidate in dental tissue engineering and bone regeneration.

Additive Manufactured Polymers in Dentistry, Current State-of-the-Art and Future Perspectives-A Review.

3D-printing application in dentistry not only enables the manufacture of patient-specific devices and tissue constructs, but also allows mass customization, as well as digital workflow, with predictable lower cost and rapid turnaround times. 4D printing also shows a good impact in dentistry, as it can produce dynamic and adaptable materials, which have proven effective in the oral environment, under its continuously changing thermal and humidity conditions. It is expected to further boost the research into producing a whole tooth, capable to harmoniously integrate with the surrounding periodontium, which represents the ultimate goal of tissue engineering in dentistry. Because of their high versatility associated with the wide variety of available materials, additive manufacturing in dentistry predominantly targets the production of polymeric constructs. The aim of this narrative review is to catch a glimpse of the current state-of-the-art of additive manufacturing in dentistry, and the future perspectives of this modern technology, focusing on the specific polymeric materials.

Highly bioactive Akermanite-Monticellite nanocomposites for bone tissue engineering: a tunable three-dimensional biological study

Emerging bioengineering approaches such as three-dimensional (3D) cell culture methods have opened new avenues for scaffolds combined with various therapeutic agents in bone tissue regeneration. In this regard, bioceramic nanocomposite compounds have been prominently studied in bone and dental tissue engineering, due to their potential for diseased bone regeneration. We synthesized in situ bioceramic nanocomposite using a combination of sol–gel and mechanical activation that the nanocomposite contained Akermanite as a major phase, and Monticellite as a minor phase. After physicochemical characterization (crystallite size of Ak and Mon were 58 and 48 nm respectively), cell viability and pertinent biological studies of the nanocomposite powder showed 1.5 times the control value. The results reported the enhanced cell viability in 2D conditions, and 3D culture with bone tissue spheroids (viability was almost 150%). Furthermore, alkaline phosphatase activity (ALP) was tested in both monolayer and spheroid conditions, and the results indicated a boosted ALP activity in 2D and 3D condition (4 and 2 times the control value respectively). Additionally, nanocomposite powder extracts promoted the expression of both the COL1 and VEGF genes in 3D cultures (5 and 1.5 times the first day value respectively), showing the extract capability in accelerating bone spheroid formation. The current proof-of-concept study showed that 3D culture technology could provide a valuable way to understand the potential of tissue engineering before undertaking costly and harsh animal studies.

Long non-coding RNA IGFBP7-AS1 accelerates the odontogenic differentiation of stem cells from human exfoliated deciduous teeth by regulating IGFBP7 expression

Stem cells from human exfoliated deciduous teeth (SHED) are attractive seed cells for dental tissue engineering. We identified the effect of the long noncoding RNA insulin-like growth factor-binding protein 7 antisense RNA 1 (lncRNA IGFBP7-AS1) in vivo and its underlying mechanism during SHED odontogenic differentiation. IGFBP7-AS1 and insulin-like growth factor-binding protein 7 (IGFBP7) were overexpressed using lentiviruses. IGFBP7 expression was knocked down with small interfering RNA. The effect of IGFBP7-AS1 in vivo was confirmed by animal experiments. The effect of IGFBP7 on SHED odontogenic differentiation was assessed with alkaline phosphatase staining, alizarin red S staining, quantitative reverse transcription-PCR, and western blotting. The relationship between IGFBP7-AS1 and IGFBP7 was confirmed by quantitative reverse transcription–PCR and western blotting. IGFBP7-AS1 promoted SHED odontogenesis in vivo, and regulated the expression of the coding gene IGFBP7 positively. Inhibiting IGFBP7 led to suppress SHED odontogenic differentiation while IGFBP7 overexpression had the opposite effect. IGFBP7-AS1 enhanced the stability of IGFBP7. IGFBP7-AS1 promoted SHED odontogenic differentiation in vivo. The underlying mechanism may involve the enhancement of IGFBP7 stability. This may provide novel potential targets for dental tissue engineering.

The influence of IGF-I and Emdogain on the behavior of dental epithelial cells in a three-dimensional scaffold model

Aim: Tooth decay and associated periodontal disease remain the most common chronic diseases in current society. In the future, individually tailored, more effective therapeutic treatment options could be provided through the application of tissue engineering and regenerative medicine and dentistry (TERMD) approaches. A relevant finding is the potential of both dental mesenchymal and dental epithelial cells to regenerate mineralized dentin and enamel tissues, respectively.Materials and Methods: In the current study, a multi-layered, bioengineered tooth bud model was assembled by combining human dental mesenchymal (hDM) and porcine dental epithelial cells (pDE). The hDM cells were seeded onto poly(epsilon-caprolactone)/ poly (lactide-co-glycolide) (PCL/PLGA) wet electro-spun scaffolds overlayed with confluent pDE cell sheet harvested from thermo-reversible tissue culture plates. The so-formed multi-layered bioengineered tooth bud was then used to study the mineralization potential of the dental cells in in vitro culture. It was hypothesized that the addition of the in media soluble factors Insulin-like growth factor 1 (IGF-I) and the extracellular matrix derivate Emdogain (EMD) would result in enhanced differentiation and mineralized dental tissue formation. Scanning Electron morphological observation was used to characterize scaffolds porosity. Histological and immunofluorescent analyses confirmed the localization of hDM cells inside the scaffold, an intact pDE cell sheet, and the presence of beta-integrin 1-positive cell-cell junctions connecting the two.Results: Scanning Electron Microscopy showed that EMD, in particular, enhanced the mineralization potential of pDE cells. qRT-PCR analyses showed that both EMD and IGF-1 significantly enhanced the expression of Ameloblastin (AMBN), reflecting pDE cell differentiation.Conclusion: In conclusion, these results proved the hypothesis that both EMD and IGF-1 should be considered for their utility in preclinical dental tissue engineering approaches.

Injectable thermosensitive chitosan/gelatin hydrogel for dental pulp stem cells proliferation and differentiation.

Introduction: Biocompatible and biodegradable scaffolds based on natural polymers such as gelatin and chitosan (CS) provide suitable microenvironments in dental tissue engineering. In the present study, we report on the synthesis of injectable thermosensitive hydrogel (PNIPAAm-g-CS copolymer/gelatin hybrid hydrogel) for osteogenic differentiation of human dental pulp stem cells (hDPSCs). Methods: The CS-g-PNIPAAm was synthesized using the reaction of carboxyl terminated PNIPAAm with CS, which was then mixed with various amounts of gelatin solution in the presence of genipin as a chemical crosslinker to gain a homogenous solution. The chemical composition and microstructures of the fabricated hydrogels were confirmed by FT-IR and SEM analysis, respectively. To evaluate the mechanical properties (e.g., storage and loss modulus of the gels), the rheological analysis was considered. Calcium deposition and ALP activity of DPSCs were carried out using alizarin red staining and ALP test. While the live/dead assay was performed to study its toxicity, the real-time PCR was conducted to investigate the osteogenic differentiation of hDPSCs cultured on prepared hydrogels. Results: The hydrogels with higher gelatin incorporation showed a slightly looser network compared to the other ones. The hydrogel with less gelatin indicates a rather higher value of G', indicating a higher elasticity due to more crosslinking reaction of amine groups of CS via a covalent bond with genipin. All the hydrogels contained viable cells with negligible dead cells, indicating the high biocompatibility of the prepared hydrogels for hDPSCs. The quantitative results of alizarin red staining displayed a significant rise in calcium deposition in hDPSCs cultured on prepared hydrogels after 21 days. Further, hDPSCs cultured on hydrogel with more gelatin displayed the most ALP activity. The expression of late osteogenic genes such as OCN and BMP-2 were respectively 6 and 4 times higher on the hydrogel with more gelatin than the control group after 21 days. Conclusion: The prepared PNIPAAm-g-CS copolymer/gelatin hybrid hydrogel presented great features (e.g., porous structure, suitable rheological behavior, and improved cell viability), and resulted in osteogenic differentiation necessary for dental tissue engineering.

Photocross-linked silk fibroin/hyaluronic acid hydrogel loaded with hDPSC for pulp regeneration

The construction of suitable biomaterials for pulp regeneration has always been a major challenge in the field of stomatology. Considering the complex and irregular anatomy of the root canal system, injectable hydrogels have received extensive attention as cell carriers in dental pulp regeneration. Here, we developed an injectable photocrosslinked methacrylylated silk fibroin (RSFMA)/methacrylylated hyaluronic acid (MeHA) composite hydrogel and characterized its physicochemical properties. The biocompatibility of encapsulated human dental pulp stem cells (hDPSCs) was subsequently investigated. With the addition of RSFMA, the pore size of the scaffolds became more regular with negligible change in porosity and exhibited excellent mechanical properties. Furthermore, the low concentration of RSFMA hydrogel in the composite hydrogel had higher cross-linking efficiency. In contrast to MeHA hydrogels, hDPSCs were encapsulated in hydrogels either in the absence or presence of high concentrations of RSFMA. The results indicated that cells in low-concentration RSFMA composite gel presented better growth ability, proliferation ability and osteogenic differentiation ability. This injectable photocrosslinked silk fibroin/hyaluronic acid hydrogel shows great potential in the field of dental pulp tissue engineering.

Direct pulp capping with autologous bone marrow derived stem cells in dogs.

Bone-marrow derived stem cells (BMSCs) can differentiate into several mesenchymal cell lines that are suitable for bone and dental tissue engineering. This study was aimed to assess the efficacy of cell therapy in direct pulp capping (DPC) of canine teeth using autologous BMSCs along with collagen/hydroxyapatite hybrid scaffold in terms of the quantity and quality of calcified bridge formation. The teeth were randomly divided into three groups of DPC with mineral trioxide aggregate (MTA), hydroxyapatite/collagen hybrid scaffold alone and BMSCs with hydroxyapatite/collagen hybrid scaffold. DPC was performed under general anesthesia in cavities prepared on the buccal surfaces of mandibular and maxillary premolars of the same dogs from which, stem cells had been isolated. All cavities were then restored with light-cure resin modified glass ionomer cement. Histomorphometric assessments after 12 weeks showed formation of dentinal bridge following DPC with BMSCs and MTA. The efficacy of MTA for calcified bridge formation following DPC was significantly higher than that of BMSCs plus hybrid scaffold. According to the present study, we concluded DPC using BMSCs and hybrid scaffold did not provide clinically noticeable results in canine patients.

Self-Crosslinkable Oxidized Alginate-Carboxymethyl Chitosan Hydrogels as an Injectable Cell Carrier for In Vitro Dental Enamel Regeneration.

Injectable hydrogels, as carriers, offer great potential to incorporate cells or growth factors for dental tissue regeneration. Notably, the development of injectable hydrogels with appropriate structures and properties has been a challenging task, leaving much to be desired in terms of cytocompatibility, antibacterial and self-healing properties, as well as the ability to support dental stem cell functions. This paper presents our study on the development of a novel self-cross-linkable hydrogel composed of oxidized alginate and carboxymethyl chitosan and its characterization as a cell carrier for dental enamel regeneration in vitro. Oxidized alginate was synthesized with 60% theoretical oxidation degree using periodate oxidation and characterized by Fourier Transform Infrared spectroscopy, proton nuclear magnetic resonance spectroscopy, and Ultraviolet-visible absorption spectroscopy. Then, hydrogels were prepared at three varying weight ratios of oxidized alginate to carboxymethyl chitosan (4:1, 3:1, and 2:1) through Schiff base reactions, which was confirmed by Fourier Transform Infrared spectroscopy. The hydrogels were characterized in terms of gelation time, swelling ratio, structure, injectability, self-healing, antibacterial properties, and in vitro characterization for enamel regeneration. The results demonstrated that, among the three hydrogels examined, the one with the highest ratio of oxidized alginate (i.e., 4:1) had the fastest gelation time and the lowest swelling ability, and that all hydrogels were formed with highly porous structures and were able to be injected through a 20-gauge needle without clogging. The injected hydrogels could be rapidly reformed with the self-healing property. The hydrogels also showed antibacterial properties against two cariogenic bacteria: Streptococcus mutans and Streptococcus sobrinus. For in vitro enamel regeneration, a dental epithelial cell line, HAT-7, was examined, demonstrating a high cell viability in the hydrogels during injection. Furthermore, HAT-7 cells encapsulated in the hydrogels showed alkaline phosphatase production and mineral deposition, as well as maintaining their round morphology, after 14 days of in vitro culture. Taken together, this study has provided evidence that the oxidized alginate-carboxymethyl chitosan hydrogels could be used as an injectable cell carrier for dental enamel tissue engineering applications.

Long Noncoding RNA IGFBP7-AS1 Promotes Odontogenesis of Stem Cells from Human Exfoliated Deciduous Teeth via the p38 MAPK Pathway.

Stem cells from human exfoliated deciduous teeth (SHED) are attractive seed cells for dental tissue engineering. Epigenetics refers to heritable changes in gene expression patterns that do not alter DNA sequences. Long noncoding RNAs (lncRNAs) are one of the main methods of epigenetic regulation and participate in cell differentiation; however, little is known regarding the role of lncRNAs during SHED odontogenic differentiation. In this study, RNA sequencing (RNA-seq) was used to obtain the expression profile of lncRNAs and mRNAs during the odontogenic differentiation of SHED. The effect of IGFBP7-AS1 on odontogenic differentiation of SHED was assessed by alkaline phosphatase (ALP) staining, alizarin red S (ARS) staining, quantitative reverse transcription PCR (qRT-PCR), Western blot, and in vivo. The level of p38 and p-p38 protein expression was examined by Western blot, and the result was verified by adding the p38 inhibitor, SB203580. The expression profiles of lncRNAs and mRNAs were identified by RNA-seq analysis, which help us to further understand the mechanism in odontogenesis epigenetically. IGFBP7-AS1 expression was increased during odontogenic differentiation on days 7 and 14. The ALP staining, ARS staining, and expression of odontogenic markers were upregulated by overexpressing IGFBP7-AS1 in vitro, whereas the expression of osteogenesis markers was not significantly changed on mRNA level. The effect of IGFBP7-AS1 was also verified in vivo. IGFBP7-AS1 could further positively regulate odontogenic differentiation through the p38 MAPK pathway. This may provide novel targets for dental tissue engineering.

Fabrication of bone-derived decellularized extracellular matrix/ceramic-based biocomposites and their osteo/odontogenic differentiation ability for dentin regeneration.

The goal of this study was to fabricate bioactive cell‐laden biocomposites supplemented with bone‐derived decellularized extracellular matrix (dECM) with calcium phosphate ceramic, and to assess the effect of the biocomponents on the osteogenic and odontogenic differentiation of human dental pulp stem cells (hDPSCs). By evaluating the rheological properties and selecting printing parameters, mechanically stable cell‐laden 3D biocomposites with high initial cell‐viability (>90%) and reasonable printability (≈0.9) were manufactured. The cytotoxicity of the biocomposites was evaluated via MTT assay and nuclei/F‐actin fluorescent analyses, while the osteo/odontogenic differentiation of the hDPSCs was assessed using histological and immunofluorescent analyses and various gene expressions. Alkaline phosphate activity and alizarin red staining results indicate that the dECM‐based biocomposites exhibit significantly higher osteogenic activities, including calcification, compared to the collagen‐based biocomposites. Furthermore, immunofluorescence images and gene expressions demonstrated upregulation of dentin matrix acidic phosphoprotein‐1 and dentin sialophosphoprotein in the dECM‐based biocomposites, indicating acceleration of the odontogenic differentiation of hDPSCs in the printed biocomposites. The hDPSC‐laden biocomposite was implanted into the subcutaneous region of mice, and the biocomposite afforded clear induction of osteo/odontogenic ectopic hard tissue formation 8 weeks post‐transplantation. From these results, we suggest that the hDPSC‐laden biocomposite is a promising biomaterial for dental tissue engineering.

Pro-angiogenic potential of a functionalized hydrogel scaffold as a secretome delivery platform: An innovative strategy for cell homing-based dental pulp tissue engineering.

Angiogenesis is a key process that provides a suitable environment for successful tissue engineering and is even more crucial in regenerative endodontic procedures, since the root canal anatomy limits the development of a vascular network supply. Thus, sustainable and accelerated vascularization of tissue-engineered dental pulp constructs remains a major challenge in cell homing approaches. This study aimed to functionalize a chitosan hydrogel scaffold (CS) as a platform loaded with secretomes of stem cells from human exfoliated deciduous teeth (SHEDs) and evaluate its bioactive function and pro-angiogenic properties. Initially, the CS was loaded with SHED secretomes (CS-S), and the release kinetics of several trophic factors were assessed. Proliferation and chemotaxis assays were performed to analyze the effect of functionalized scaffold on stem cells from apical papilla (SCAPs) and the angiogenic potential was analyzed through the Matrigel tube formation assay with co-cultured of human umbilical vein endothelial cells and SCAPs. SHEDs and SCAPs expressed typical levels of mesenchymal stem cell surface markers. CS-S was able to release the trophic factors in a sustained manner, but each factor has its own release kinetics. The CS-S group showed a significantly higher proliferation rate, accelerated the chemotaxis, and higher capacity to form vascular-like structures. CS-S provided a sustained and controlled release of trophic factors, which, in turn, improved proliferation, chemotaxis and all angiogenesis parameters in the co-culture. Thus, the functionalization of chitosan scaffolds loaded with secretomes is a promising platform for cell homing-based tissue engineering.

Molecular Biological Comparison of Dental Pulp- and Apical Papilla-Derived Stem Cells

Both the dental pulp and the apical papilla represent a promising source of mesenchymal stem cells for regenerative endodontic protocols. The aim of this study was to outline molecular biological conformities and differences between dental pulp stem cells (DPSC) and stem cells from the apical papilla (SCAP). Thus, cells were isolated from the pulp and the apical papilla of an extracted molar and analyzed for mesenchymal stem cell markers as well as multi-lineage differentiation. During induced osteogenic differentiation, viability, proliferation, and wound healing assays were performed, and secreted signaling molecules were quantified by enzyme-linked immunosorbent assays (ELISA). Transcriptome-wide gene expression was profiled by microarrays and validated by quantitative reverse transcription PCR (qRT-PCR). Gene regulation was evaluated in the context of culture parameters and functionality. Both cell types expressed mesenchymal stem cell markers and were able to enter various lineages. DPSC and SCAP showed no significant differences in cell viability, proliferation, or migration; however, variations were observed in the profile of secreted molecules. Transcriptome analysis revealed the most significant gene regulation during the differentiation period, and 13 biomarkers were identified whose regulation was essential for both cell types. DPSC and SCAP share many features and their differentiation follows similar patterns. From a molecular biological perspective, both seem to be equally suitable for dental pulp tissue engineering.

Isolation of Endogenous TGF-β1 from Root Canals for Pulp Tissue Engineering: A Translational Study.

Simple SummaryTissue engineering of the dental pulp has been a goal of dental research for years. In this translational study, a chairside protocol is designed using endogenous dentin matrix proteins as signaling molecules for pulp regeneration. These bioactive molecules can be isolated from root canals by ultrasonic-activated irrigation, further processed chairside, and mixed with a hydrogel. The scaffold material is to be injected into the root canal and effect cell homing, i.e., allowing stem cells from the periapical space to migrate into the root canal. The aim of this innovative approach is the formation of an innervated and vascularized connective tissue that resembles the pulp in form and function.Cell homing for dental pulp tissue engineering has been advocated as a feasible approach to regenerate dental pulp in a clinical setting. In order to develop a translational protocol for clinical application, we wanted to determine the effects of disinfectants on the availability of growth factors from the root canal, the amount that can be obtained in this context, and whether they can be processed for use in tissue engineering procedures. The extraction of growth factors should also be confirmed in a clinical setting. Root canals were prepared in 36 extracted mature teeth, and the amount of TGF-β1 in solution was quantified after different irrigation protocols (sodium hypochlorite, chlorhexidine) and after intracanal medication (calcium hydroxide). Centrifugal filters with a cut-off of 10,000 Da and 3000 Da were used for efficient concentration, and volumes and amounts of retained TGF-β1 were measured at different time points. During conventional endodontic treatment, ethylenediaminotetraacetic acid (EDTA) solution was collected after ultrasonic activation from the root canals of mature teeth of 38 patients, and growth factor content was quantified via enzyme-linked immunosorbent assay (ELISA). Irrigation with sodium hypochlorite reduced TGF-β1 release into EDTA. This effect was partially reversed by canal enlargement after the use of sodium hypochlorite and by subsequent use of calcium hydroxide. A few minutes of centrifugation with a cut-off of 10,000 Da reduced the initial volume of the irrigant by 90% and led to a continuous increase in concentration to the same extent. Furthermore, TGF-β1 was obtained from root canals of mature teeth during endodontic treatment in quantities that have been shown to elicit desirable cellular responses in a subsequent clinical application. A mixture with a suitable scaffold material and injection into the root canal has the potential to promote dental pulp regeneration.

Preparation, Physicochemical Assessment and the Antimicrobial Action of Hydroxyapatite–Gelatin/Curcumin Nanofibrous Composites as a Dental Biomaterial

In this study, we prepared and evaluated hydroxyapatite–gelatin/curcumin nanofibrous composites and determined their antimicrobial effects against Escherichia coli, Staphylococcus aureus, and Streptococcus mutans. Hydroxyapatite–gelatin/curcumin nanofibrous composites were prepared by the electrospinning method. The prepared nanocomposites were then subjected to physicochemical studies by the light scattering method for their particle size, Fourier transmission infrared spectroscopy (FTIR) to identify their functional groups, X-ray diffraction (XRD) to study their crystallinity, and scanning electron microscopy (SEM) to study their morphology. For the microbial evaluation of nanocomposites, the disk diffusion method was used against Streptococcus mutans, Staphylococcus aureus, and Escherichia coli. The results showed that the nanofibers were uniform in shape without any bead (structural defects). The release pattern of curcumin from the nanocomposite was a two-stage release, 60% of which was released in the first two days and the rest being slowly released until the 14th day. The results of the microbial evaluations showed that the nanocomposites had significant antimicrobial effects against all bacteria (p = 0.0086). It seems that these nanocomposites can be used in dental tissue engineering or as other dental materials. Also, according to the appropriate microbial results, these plant antimicrobials can be used instead of chemical antimicrobials, or along with them, to reduce bacterial resistance.

In vitro evaluation of periapical lesion-derived stem cells for dental pulp tissue engineering.

Dental pulp tissue engineering is a promising alternative treatment for pulpitis and periapical periodontitis, and dental pulp stem cells (DPSCs) are considered to be the gold standard for dental seed cell research. Periapical lesions harbor mesenchymal stem cells with the capacity for self‐renewal and multilineage differentiation. However, it remains unknown whether these periapical lesion‐derived stem cells (PLDSCs) are suitable for dental pulp tissue engineering. To investigate this possibility, PLDSCs and DPSCs were isolated using the tissue outgrowth method and cultured under identical conditions. We then performed in vitro experiments to investigate their biological characteristics. Our results indicate that PLDSCs proliferate actively in vitro and exhibit similar morphology, immunophenotype and multilineage differentiation ability as DPSCs. Simultaneously, PLDSCs exhibit stronger migrative ability and express more vascular endothelial growth factor and glial cell line‐derived neurotrophic factor than DPSCs, and PLDSC‐derived conditioned medium was more effective in tube formation assay. The mRNA expression levels of immunomodulatory genes HLA‐G, IDO and ICAM‐1 were also higher in PLDSCs. However, regarding osteo/odontogenic differentiation, PLDSCs showed weaker alkaline phosphatase staining and lower calcified nodule formation compared to DPSCs, as well as lower expression of ALP, RUNX2 and DSPP, as confirmed by a quantitative RT‐PCR. The osteo/odontogenic protein expression levels of DSPP, RUNX2, DMP1 and SP7 were also higher in DPSCs. The present study demonstrates that PLDSCs demonstrate potential use as seed cells for dental pulp regeneration, especially for achieving enhanced neurovascularization.

Schwann cell-derived EVs facilitate dental pulp regeneration through endogenous stem cell recruitment via SDF-1/CXCR4 axis

The dental pulp is critical for physiological vitality of the tooth, and dental pulp regeneration has great potential for rebuilding live pulp tissue after pulp disease. Schwann cells (SCs) play a critical role in the support, maintenance, and regeneration of nerve fibers in dental pulp. Extracellular vesicles (EVs), which possess cell homing and tissue repair potential, derived from SCs (SC-EVs), can regulate dental mesenchymal stem cells (MSCs) proliferation, multipotency, and self-renewal. However, the role of SC-EVs in dental pulp tissue regeneration remains unclear. To address this question, we treated dental pulp stem cells (DPSCs) and bone marrow stem cells (BMSCs) with SC-EVs, and the results showed an obvious increase in the proliferation, migration, and osteogenic differentiation of both cell types. SC-EVs also promoted neurite outgrowth and neuron migration of rat dorsal root ganglia, as well as vessel formation in vitro. In an in vivo model of subcutaneous, SC-EVs enhanced the recruitment of endogenous vascular endothelioid-like cells and MSCs, and promoted the formation of a pulpo-dentinal complex-like structure. Finally, mass spectrometry analyses and western blot revealed that stromal cell-derived factor 1 (SDF-1, also known as CXCL12) plays a dominant role in SC-EVs. Together, these data suggest that SC-EVs successfully recruit endogenous stem cells to promote dental pulp regeneration. Our results provide a cell-free strategy for pulp regeneration that avoids the risks associated with stem cell transplantation. Statement of SignificanceDental pulp is vulnerable to infections resulting from dental care, trauma, and multiple restorations, with such infections resulting in pulpitis and pulp necrosis. The current endodontic treatment of irreversible pulp disease cannot restore the function of dental pulp and tissue engineering strategies using cell-based approaches are limited by several disadvantages, including immune rejection and limited cell sources. In this study, we found that schwann cells-derived EVs facilitated dental pulp regeneration through endogenous stem cells recruitment via SDF-1/CXCR4 axis without exogenous cell transplantation. We believe that our study makes a significant contribution to describe a cell-free strategy to promote dental pulp regeneration.