Dental Pulp-like Tissue Research Articles

Correction: Ferroni et al. A Hyaluronan-Based Scaffold for the in Vitro Construction of Dental Pulp-Like Tissue. Int. J. Mol. Sci. 2015, 16, 4666–4681

In the original publication [...]

Regenerative Endodontics -Present Practice to Promising Prospects: A Literature Review

Regenerative endodontics is an innovative field within dentistry that aims to restore damaged dental pulp using stem cells, tissue engineering techniques and biocompatible materials. This article provides an overview of the current trends in regenerative endodontics, highlighting recent research findings. Stem cells obtained from different sources like dental pulp, bone marrow and adipose tissue possess the ability to differentiate into cells similar to dental pulp and facilitate the regeneration of tissues. The use of biocompatible substances, such as Mineral Trioxide Aggregate (MTA) biodentine, offers an ideal setting for tissue growth and healing. Dental scaffolds, made from biodegradable materials, support the development of new blood vessels, nerve fibers and dental pulp-like tissues. Growth factors, like PDGF and TGF-β, are incredibly important in stimulating cell growth and specialization. Researchers and clinical trials are currently working on improving regenerative endodontic procedures, with promising results seen in terms of pulp regeneration and root development. By embracing these trends, dental professionals can offer innovative solutions for restoring damaged dental pulp and preserving natural teeth. The present article, is an overview of regenerative endodontics, exploring the current trends that are shaping the future of this field.

Allogeneic Bone Marrow Mesenchymal Stromal Cell Transplantation Induces Dentin Pulp Complex-like Formation in Immature Teeth with Pulp Necrosis and Apical Periodontitis

IntroductionDental pulp regeneration is challenging in endodontics. Cellular therapy is an alternative approach to induce dental pulp regeneration. Mesenchymal stromal cells (MSCs) have the capacity to induce dental pulp-like tissue formation. In this study, we evaluated the capacity of allogeneic bone marrow MSCs (BM-MSCs) to regenerate pulp following necrosis and apical periodontitis in children's permanent immature apex teeth. MethodsPatients aged 8 to 12 years with pulp necrosis and apical periodontitis were evaluated. The study included 15 teeth (13 incisors and 2 molars) from 14 patients (8 boys and 6 girls). Radiographic evaluation showed periapical radiolucency and immature apex teeth. There was no response to cold or electric pulp testing. The root canal of each tooth was cleaned, shaped, and Ca(OH)2 used as an interappointment medication. Cryopreserved allogeneic BM-MSCs were thawed, expanded, incorporated into preclotted platelet-rich plasma, and implanted into the tooth's pulp cavity. They were sealed with bioceramic cement and composite. Sensibility, apical foramen, calcium deposits within the root canal, and resolution of periapical lesions were evaluated in each tooth over the following 12 months. ResultsBased on 9 variables established for dental pulp-like tissue regeneration, all MSC-treated teeth showed evidence of successful regeneration. Clinical and radiographic evaluation of the treated teeth showed periapical lesion healing, sensitivity to cold and electricity, decreased width of the apical foramen, and mineralization within the canal space. ConclusionsTransplantation of allogeneic MSCs induces the formation of dental pulp-like tissue in permanent immature apex teeth with pulp necrosis and apical periodontitis. Implant of MSCs constitutes a potential therapy in regenerative endodontics in pediatric dentistry. Future studies incorporating a larger sample size may confirm these results.

Inhibition of soluble epoxide hydrolase enhances the dentin-pulp complex regeneration mediated by crosstalk between vascular endothelial cells and dental pulp stem cells

BackgroundRevascularization and restoration of normal pulp-dentin complex are important for tissue-engineered pulp regeneration. Recently, a unique periodontal tip-like endothelial cells subtype (POTCs) specialized to dentinogenesis was identified. We have confirmed that TPPU, a soluble epoxide hydrolase (sEH) inhibitor targeting epoxyeicosatrienoic acids (EETs) metabolism, promotes bone growth and regeneration by angiogenesis and osteogenesis coupling. We hypothesized that TPPU could also promote revascularization and induce POTCs to contribute to pulp-dentin complex regeneration. Here, we in vitro and in vivo characterized the potential effect of TPPU on the coupling of angiogenesis and odontogenesis and investigated the relevant mechanism, providing new ideas for pulp-dentin regeneration by targeting sEH.MethodsIn vitro effects of TPPU on the proliferation, migration, and angiogenesis of dental pulp stem cells (DPSCs), human umbilical vein endothelial cells (HUVECs) and cocultured DPSCs and HUVECs were detected using cell counting kit 8 (CCK8) assay, wound healing, transwell, tube formation and RT-qPCR. In vivo, Matrigel plug assay was performed to outline the roles of TPPU in revascularization and survival of grafts. Then we characterized the VEGFR2 + POTCs around odontoblast layer in the molar of pups from C57BL/6 female mice gavaged with TPPU. Finally, the root segments with DPSCs mixed with Matrigel were implanted subcutaneously in BALB/c nude mice treated with TPPU and the root grafts were isolated for histological staining.ResultsIn vitro, TPPU significantly promoted the migration and tube formation capability of cocultured DPSCs and HUVECs. ALP and ARS staining and RT-qPCR showed that TPPU promoted the osteogenic and odontogenic differentiation of cultured cells, treatment with an anti-TGF-β blocking antibody abrogated this effect. Knockdown of HIF-1α in HUVECs significantly reversed the effect of TPPU on the expression of angiogenesis, osteogenesis and odontogenesis-related genes in cocultured cells. Matrigel plug assay showed that TPPU increased VEGF/VEGFR2-expressed cells in transplanted grafts. TPPU contributed to angiogenic-odontogenic coupling featured by increased VEGFR2 + POTCs and odontoblast maturation during early dentinogenesis in molar of newborn pups from C57BL/6 female mice gavaged with TPPU. TPPU induced more dental pulp-like tissue with more vessels and collagen fibers in transplanted root segment.ConclusionsTPPU promotes revascularization of dental pulp regeneration by enhancing migration and angiogenesis of HUVECs, and improves odontogenic differentiation of DPSCs by TGF-β. TPPU boosts the angiogenic–odontogenic coupling by enhancing VEGFR2 + POTCs meditated odontoblast maturation partly via upregulating HIF-1α, which contributes to increasing pulp-dentin complex for tissue-engineered pulp regeneration.

The effect of human umbilical cord mesenchymal stem cells combined with concentrated growth factor on repairing necrotic pulp caused by dental caries.

This study investigated the impact of combining human umbilical cord mesenchymal stem cells (hUC-MSCs) with concentrated growth factor (CGF) on regenerating necrotic pulp. Ten-month-old male Bama miniature pigs were divided into control and caries groups. The experimental teeth were randomly divided into three groups: caries untreated, Ca(OH)2, and engineering dental pulp-like tissue (EDPT). hUC-MSCs and CGF scaffold were combined to construct EDPT, and the histological structure was observed. Odontoblasts and dental pulp cells were counted in each group. The results showed that hUC-MSCs adhered firmly to the porous mesh CGF scaffold, grew vigorously, and stretched sufficiently. In the EDPT group, odontoblasts in the root canal were arranged neatly, and predentin was formed. The odontoblast and dental pulp cell counts in the EDPT group were statistically significant compared to the caries untreated and Ca(OH)2 groups. The hUC-MSCs-CGF could successfully repair necrotic pulp in animals with dental caries.

Three-Dimensional Culture of Human Dental Pulp Tissue: A Preliminary Experimental In Vitro Model for Regenerative Endodontic Procedures

Introduction: Dental pulp regeneration is fundamental in dentistry and endodontics; however, few in vitro experimental models are available to study its biological process. This study aimed to develop a three-dimensional (3D) culture model of human dental pulp-like tissue mimicking the possible complexity of human pulp tissue. This new and innovative human-like tissue model could be used for testing drugs and materials, particularly those involved in regenerative endodontics. Methods: Vital pulp tissue samples were obtained from human third molars (n = 4) immediately after extraction and cultured in a 3D fibrin matrix to create a sustainable ex vivo experimental model. The angiogenesis degrees and the nitric oxide levels were evaluated following the culture of pulp-like tissues in the fibrin matrix for 21 days. The expression of Transforming growth factor- beta (TGF-β1), TGF-β2, TGF-β3, and their relevant receptors, tumor necrosis factor (TNF) and vascular endothelial growth factor A (VEGFA) was evaluated using a reverse transcription polymerase chain reaction (RT-PCR) method. Results: Pulp tissue angiogenesis was initiated, and completed on days 7 and 21, and pulp-like tissue cells expressed TGF-β1, TGF-β2, TGF-β3, and their relevant receptors, TNF and VEGFA. Conclusion: This model provided a precise observation of dental pulp angiogenesis at early stages.

Wireless electrical stimulation generated by piezoelectric nanomaterial promotes the dental pulp regeneration via regulating mitochondrial Ca2+/PKA signaling pathway

The induction of dental pulp stem cells (DPSCs) into odontogenic differentiation is a cutting-edge method of dental pulp regeneration treatment. However, it remains a challenge to develop biomaterials and therapies that can induce odontogenic differentiation. Here, we propose a wireless electrical stimulation strategy to induce DPSCs odontogenic differentiation via K0.5Na0.5NbO3 piezoelectric nanoparticles (KNN) and polarized KNN (PKNN), which can be endocytosed by DPSCs. In vitro, several odontogenic differentiation indexes were also increased in DPSCs treated with KNN and PKNN, and the increase was more obvious in the PKNN group. Intracellular wireless electrical field promoted mitochondrial calcium concentration via mitochondrial calcium unidirectional transporter (MCU), increased the production of adenosine triphosphate (ATP), and induced odontogenic differentiation through the activation of the cAMP-PKA signaling pathway. In vivo, dental pulp-like tissue was induced by electrical stimulation wirelessly with KNN and PKNN, which was more clinically friendly compared with the wired device, and the induction was more obvious in the PKNN group consistent with in vitro experiments. In conclusion, this work demonstrates the potential of PKNN as an effective stimulus that can induce odontogenic differentiation of DPSCs and be applied to dental pulp regeneration treatment.

Decellularized rat submandibular gland as an alternative scaffold for dental pulp regeneration.

Introduction: Decellularized extracellular matrix has been recognized as an optimal scaffold for dental pulp regeneration. However, the limited amount of native dental pulp tissue restricts its clinical applications. The submandibular gland shares some basic extracellular matrix components and characteristics with dental pulp. However, whether decellularized submandibular gland extracellular matrix (DSMG) can be used as an alternative scaffold for dental pulp regenerative medicine is unclear. Methods: Thus, we successfully decellularized the whole rat submandibular gland and human dental pulp, and then conducted in vitro and in vivo studies to compare the properties of these two scaffolds for dental pulp regeneration. Results: Our results showed that extracellular matrix of the submandibular gland had great similarities in structure and composition with that of dental pulp. Furthermore, it was confirmed that the DSMG could support adhesion and proliferation of dental pulp stem cells in vitro. In vivo findings revealed that implanted cell-seeded DSMG formed a vascularized dental pulp-like tissue and expressed markers involved in dentinogenesis and angiogenesis. Discussion: In summary, we introduced a novel accessible biological scaffold and validated its effectiveness as an extracellular matrix-based tissue engineering scaffold for dental pulp regenerative therapy.

The role of different macrophages-derived conditioned media in dental pulp tissue regeneration

Macrophages have been reported to play important roles in tissue repair and regeneration. While it is known that macrophages are present in the dental pulp, their role in dental pulp regeneration is not fully understood. In the present study, we investigated the effects of different phenotype macrophages conditioned medium on the cellular behaviors of hDPSCs and their extracellular matrix (cell sheets) in vitro. Moreover, twenty-four root fragments inserted with cell sheets cultured with different conditioned media were placed into the back subcutaneous space of 6–8-week-old male BALB/c nude mouse. The regenerated tissues in the root fragments were assessed via histologic analysis after 8 weeks of transplantation. M2 macrophages could promote the proliferation, migration, and osteogenic differentiation of hDPSCs. Dental pulp-like tissue with an odontoblast-like layer lining the dentinal surface and well-arranged collagen fibers was harvested in root fragment combined with M2 conditioned medium cultured cell sheet, whereas a large amount of calcium salt deposition and disorganization of collagen fibers were observed in root fragments combined with M1 conditioned medium cultured cell sheet. Therefore, promoting the transformation of M1 into M2 macrophage in dental pulp tissue regeneration may be a potential way for dental pulp regeneration via functional healing.

Salidroside promotes the osteogenic and odontogenic differentiation of human dental pulp stem cells through the BMP signaling pathway.

Regenerative endodontics, as an alternative approach, aims to regenerate dental pulp-like tissues and is garnering the attention of clinical dentists. This is due to its reported biological benefits for dental therapeutics. Stem cells and their microenvironment serve an important role in the process of pulp regeneration. Regulation of the stem cell microenvironment and the directed differentiation of stem cells is becoming a topic of intensive research. Salidroside (SAL) is extracted from the root of Rhodiola rosea and it has been reported that SAL exerts antiaging, neuroprotective, hepatoprotective, cardioprotective and anticancer effects. However, the ability of SAL to regulate the osteo/odontogenic differentiation of hDPSCs remains to be elucidated. In the present study, the effect of SAL on the proliferation and osteogenic/odontogenic differentiation of human dental pulp stem cells (hDPSCs) was investigated. This was achieved by performing CCK-8 ARS staining assay, reverse transcription-quantitative PCR to detect mRNA of ALP, OSX, RUNX2, OCN, DSPP and BSP, western blotting to detect the protein of MAPK, Smad1/5/8, OSX, RUNX2, BSP and GAPDH and immunofluorescence assays to detect DSPP. The results indicated that SAL promoted the cell viability and the osteogenic/odontogenic differentiation of hDPSCs whilst increasing the expression of genes associated with osteogenic/odontogenic differentiation by ARS staining assay. In addition, SAL promoted osteogenic and odontogenic differentiation by activating the phosphorylation of Smad1/5/8. Collectively, these findings suggest that SAL promoted the osteogenic and odontogenic differentiation of hDPSCs activating the BMP signaling pathway.

Human Umbilical Cord Mesenchymal Stem Cell Differentiation Into Odontoblast-Like Cells and Endothelial Cells: A Potential Cell Source for Dental Pulp Tissue Engineering.

ObjectivesDental pulp regeneration is considered an ideal approach for treating dental pulp disease. Because pulp is composed of various cells, determining the proper seed cells is critical. We explored the potential of human umbilical cord mesenchymal stem cells (hUCMSCs) as seed cells for dental pulp regeneration.MethodsLiquid extract of human treated dentin matrix (LE-TDM) was acquired to culture hUCMSCs. Odontoblast-specific markers were detected by western blot, qRT-PCR, and immunofluorescence assays. Endothelial differentiation of hUCMSCs was examined according to VEGF induction by western blot, qRT-PCR, and Matrigel assays. hUCMSCs and VEGF-induced hUCMSCs (V-hUCMSCs) were also cocultured in vivo for the Matrigel plug assay and in vitro for RNA-sequencing (RNA-seq). Finally, encapsulated mono-cultured hUCMSCs or cocultured hUCMSCs and V-hUCMSCs in scaffolds were injected into the root segments and transplanted into immunodeficient mice for dental pulp regeneration.ResultsUnder LE-TDM induction, hUCMSCs expressed specific odontoblast markers (DSPP, DMP-1, DSP). Under VEGF induction, hUCMSCs expressed functional endothelial markers (CD31, eNOs, vWF). In vivo, the Matrigel plug assay indicated that cocultured hUCMSCs and V-hUCMSCs formed extensive vessel-like structures. RNA-seq results indicated that cocultured V-hUCMSCs exhibited high Hif-1 signaling pathway activity. Both the hUCMSCs mono-culture and coculture groups showed pulp-like tissue regeneration. The cocultured group showed more extracellular matrix and vascularization than the mono-cultured group in vivo.ConclusionhUCMSCs can differentiate into odontoblast-like cells and functional endothelial cells. Cocultured hUCMSCs and V-hUCMSCs formed vessel-like structures and regenerated dental pulp-like tissue. Therefore, hUCMSCs can be used as an alternative seed cell source for angiogenesis and dental pulp regeneration.

Exosomes Derived from Stem Cells from the Apical Papilla Promote Dentine-Pulp Complex Regeneration by Inducing Specific Dentinogenesis.

Regenerative endodontic procedures (REPs) are a new option for the treatment of dental pulp or periapical diseases in permanent teeth with open apices. Histologically, the new tissues formed in the root canal after REPs are mainly cementum- or bone-like mineralised tissues, but not the real dentine-pulp complex. Therefore, how to promote dentine-pulp complex regeneration and improve the clinical effects of REPs has become a prominent research topic. Stem cells from apical papilla (SCAP) are derived from the dental papilla that can differentiate into primary odontoblasts and dental pulp cells that produce root dentine and dental pulp. Exosomes are the key regulator for the paracrine activity of stem cells and can influence the function of recipient cells. In this study, SCAP-derived exosomes (SCAP-Exo) were introduced into the root fragment containing bone marrow mesenchymal stem cells (BMMSCs) and transplanted subcutaneously into immunodeficient mice. We observed that dental pulp-like tissues were present and the newly formed dentine was deposited onto the existing dentine in the root canal. Afterwards, the effects of SCAP-Exo on the dentinogenesis of BMMSCs were elucidated in vitro. We found that the gene and protein expression of dentine sialophosphoprotein and mineralised nodule formation in BMMSCs treated with SCAP-Exo were significantly increased. In summary, SCAP-Exo were endocytosed by BMMSCs and obviously improved their specific dentinogenesis. The use of exosomes derived from dental stem cells could comprise a potential therapeutic approach for dentine-pulp complex regeneration in REPs.

Cotransplantation of human umbilical cord mesenchymal stem cells and endothelial cells for angiogenesis and pulp regeneration in vivo

AimsTo explored the potential of human umbilical cord mesenchymal stem cells (hUCMSCs) as seed cells for dental pulp regeneration and the possibility of cotransplantation hUCMSCs and endothelial cells (ECs) for angiogenesis and pulp regeneration in vivo. Materials and methodshUCMSCs and human umbilical vein endothelial cells (HUVECs) were cocultured for matrigel angiogenesis assay in vitro and Matrigel plug assay in vivo. Next, we used the transwell coculture system to coculture hUCMSCs and HUVECs in vitro for RNA- sequencing (RNA-seq). Last, encapsulated hUCMSCs and HUVECs in scaffolds were injected into the root segments, and transplanted into immunodeficient mice for dental pulp regeneration. Key findingsIn vitro Matrigel angiogenesis assay and in vivo Matrigel plug assay indicated that cocultured hUCMSCs and HUVECs promote vascular formation of HUVECs, especially in 1:5 (hUCMSCs:HUVECs) coculture group. The RNA-seq result indicated that cocultured HUVECs exhibited high Hif-1 signaling pathway activity. We performed the cell transfection assay to knock down HIF1A-AS2 in HUVECs and then coculture with hUCMSCs, and the expression of VEGFA, HIF1A and PECAM1 were reduced. In pulp regeneration assay, Cotransplantation of hUCMSCs and HUVECs (1,5) group showed pulp-like tissue regeneration. SignificanceCocultured hUCMSCs and HUVECs can promote vascular formation of HUVECs, and the optimal coculture ration is 1:5 (hUCMSCs:HUVECs). hUCMSCs promote angiogenesis of HUVECs through the long noncoding RNA HIF1A-AS2-activation of the Hif-1 signaling pathway. Cotransplantation of hUCMSCs and HUVECs can regenerate dental pulp-like tissue in vivo.

3D Printing of Stem Cell Responsive Ionically-Crosslinked Polyethylene Glycol Diacrylate/ Alginate Composite Hydrogels Loaded with Basic Fibroblast Growth Factor for Dental Pulp Tissue Engineering: A Preclinical Evaluation in Animal Model

The main aim of this study was to develop a 3D printed stem cell-responsive polyethylene glycol diacrylate (PEGDA) and sodium alginate (SA) composite hydrogel system (PEGDA/SA) loaded with basic fibroblast growth factor (bFGF) suitable for dental pulp tissue engineering. The PEGDA/SA loaded with bFGF (PEGDA/SA-bFGF) at different concentration ratios were 3D printed via stereolithography (SLA) under optimal processing conditions, followed by ionic crosslinking. The morphological and porous structure of the 3D printed PEGDA/SA-bFGF constructs and release profile of the bFGF were analyzed. The cellular compatibility of the hydrogel constructs was evaluated using human dental pulp stem cells (hDPSCs)in vitro. Subsequently, biocompatibility and tissue regenerative potential of the hydrogel constructs were evaluated in immunodeficient mice model. The in vitro cell culture results of PEGDA/SA-bFGF constructs exhibited high cell compatibility and supported cell proliferation, irrespective of their concentration ratios. Thein vivo animal experiment results showed that the hDPSCs-laden PEGDA/SA (PEGDA/SA-hDPSCs) group failed to form a good pulp structure even at various concentration ratios. When the mass ratio of PEGDA/SA was 25:1, hDPSCs-laden PEGDA/SA-bFGF (PEGDA/SA-bFGF-hDPSCs) group also failed to form dental pulp-like tissue but a partial loose connective tissue formation was observed. However, when the mass ratio of PEGDA/SA reduces to 20:1 ~ 15:1, the PEGDA/SA-bFGF-hDPSCs group tends to form a well-organized pulp structure post 4 weeks implantation, thus highlighting the potential of this particular hydrogel scaffolding system for dental pulp tissue regenerative applications.

Dental pulp regeneration via cell homing.

The typical treatment for irreversibly inflamed/necrotic pulp tissue is root canal treatment. As an alternative approach, regenerative endodontics aims to regenerate dental pulp-like tissues using two possible strategies: cell transplantation and cell homing. The former requires exogenously transplanted stem cells, complex procedures and high costs; the latter employs the host's endogenous cells to achieve tissue repair/regeneration, which is more clinically translatable. This systematic review examines cell homing for dental pulp regeneration, selecting articles on invitro experiments, invivo ectopic transplantation models and insitu pulp revascularization. MEDLINE/PubMed and Scopus databases were electronically searched for articles without limits in publication date. Two reviewers independently screened and included papers according to the predefined selection criteria. The electronic searches identified 46 studies. After title, abstract and full-text examination, 10 articles met the inclusion criteria. In vitro data highlighted that multiple cytokines have the capacity to induce migration, proliferation and differentiation of dental pulp stem/progenitor cells. The majority of the invivo studies obtained regenerated connective pulp-like tissues with neovascularization. In some cases, the samples showed new innervation and new dentine deposition. The insitu pulp revascularization regenerated intracanal pulp-like tissues with neovascularization, innervation and dentine formation. Cell homing strategies for pulp regeneration need further understanding and improvement if they are to become a reliable and effective approach in endodontics. Nevertheless, cell homing currently represents the most clinically viable pathway for dental pulp regeneration.

Cell-derived micro-environment helps dental pulp stem cells promote dental pulp regeneration.

The function of the dental pulp is closely connected to the extracellular matrix (ECM) structure, and ECM has received significant attention due to its biological functions for regulating cells. As such, the interaction between the ECM niche and cells is worth exploring for potential clinical uses. In this study, dental pulp stem cell (DPSC)-derived ECM (DPM) was prepared through cell culture and decellularization to function as the cell niche, and changes in DPSC behaviour and histological analysis of dental pulp tissue regeneration were evaluated following the DPM culture. DPM promoted the replication of DPSCs and exhibited retention of their mineralization. Then, the DPM-based culture strategy under odontogenic culture medium was further investigated, and the mineralization-related markers showed that DPSCs were regulated towards odontogenic differentiation. Dental pulp-like tissue with well-arranged ECM was harvested after a 2-month subcutaneous implantation in nude mice with DPM application. Additionally, DPSCs cultured on the plastic culture surface showed the up-regulation of mineralization makers in vitro, but there was a disorder in matrix formation and mineralization when the cells were cultured in vivo. DPM-based cultivation could serve as a cell niche and modulate DPSC behaviour, and this method also provided an alternative to harvest tissue-specific ECM and provided a strategy for ECM-cell interaction.

Suitability of Different Natural and Synthetic Biomaterials for Dental Pulp Tissue Engineering.

Dental pulp tissue engineering is possible after insertion of pulpal stem cells combined with a scaffold into empty root canals. Commonly used biomaterials are collagen or poly(lactic) acid, which are either difficult to modify or to insert into such a narrow space. New hydrogel scaffolds with bioactive, specifically tailored functions could optimize the conditions for this approach. Different synthetic and natural hydrogels were tested for their suitability to engineer dental pulp. Two functionalized modifications of polyethylene glycol were developed in this study and compared to a self-assembling peptide, as well as to collagen and fibrin. Cell viability of dental pulp stem cells in test materials was assessed over two weeks. Cells in selected test materials laden with dentin-derived growth factors were inserted into human tooth roots and implanted subcutaneously into immunocompromised mice. In vitro cell culture exhibited distinct differences between scaffold types, where viability was significantly higher in natural compared to synthetic materials. In vivo experiments showed considerable differences regarding scaffold degradation, soft tissue formation, vascularization, and odontoblast-like cell differentiation. Fibrin appeared most suitable to enable generation of a pulp-like tissue and differentiation of cells into odontoblasts at the cell-dentin interface. In conclusion, natural materials, especially fibrin, proved to be superior compared to synthetic scaffolds regarding cell viability and dental pulp-like tissue formation.

A Novel Strategy to Engineer Pre-Vascularized Full-Length Dental Pulp-like Tissue Constructs

The requirement for immediate vascularization of engineered dental pulp poses a major hurdle towards successful implementation of pulp regeneration as an effective therapeutic strategy for root canal therapy, especially in adult teeth. Here, we demonstrate a novel strategy to engineer pre-vascularized, cell-laden hydrogel pulp-like tissue constructs in full-length root canals for dental pulp regeneration. We utilized gelatin methacryloyl (GelMA) hydrogels with tunable physical and mechanical properties to determine the microenvironmental conditions (microstructure, degradation, swelling and elastic modulus) that enhanced viability, spreading and proliferation of encapsulated odontoblast-like cells (OD21), and the formation of endothelial monolayers by endothelial colony forming cells (ECFCs). GelMA hydrogels with higher polymer concentration (15% w/v) and stiffness enhanced OD21 cell viability, spreading and proliferation, as well as endothelial cell spreading and monolayer formation. We then fabricated pre-vascularized, full-length, dental pulp-like tissue constructs by dispensing OD21 cell-laden GelMA hydrogel prepolymer in root canals of extracted teeth and fabricating 500 µm channels throughout the root canals. ECFCs seeded into the microchannels successfully formed monolayers and underwent angiogenic sprouting within 7 days in culture. In summary, the proposed approach is a simple and effective strategy for engineering of pre-vascularized dental pulp constructs offering potentially beneficial translational outcomes.

Exosomes as biomimetic tools for stem cell differentiation: Applications in dental pulp tissue regeneration

Achieving and maintaining safe and reliable lineage specific differentiation of stem cells is important for clinical translation of tissue engineering strategies. In an effort to circumvent the multitude of problems arising from the usage of growth factors and growth factor delivery systems, we have explored the use of exosomes as biomimetic tools to induce stem cell differentiation. Working on the hypothesis that cell-type specific exosomes can trigger lineage-specific differentiation of stem cells, we have evaluated the potential of exosomes derived from dental pulp cells cultured on under growth and odontogenic differentiation conditions to induce odontogenic differentiation of naïve human dental pulp stem cells (DPSCs) and human bone marrow derived stromal cells (HMSCs) invitro and invivo. Results indicate that the exosomes can bind to matrix proteins such as type I collagen and fibronectin enabling them to be tethered to biomaterials. The exosomes are endocytosed by both DPSCs and HMSCs in a dose-dependent and saturable manner via the caveolar endocytic mechanism and trigger the P38 mitogen activated protein kinase (MAPK) pathway. In addition, the exosomes also trigger the increased expression of genes required for odontogenic differentiation. When tested invivo in a tooth root slice model with DPSCs, the exosomes triggered regeneration of dental pulp-like tissue. However, our results indicate that exosomes isolated under odontogenic conditions are better inducers of stem cell differentiation and tissue regeneration. Overall, our results highlight the potential exosomes as biomimetic tools to induce lineage specific differentiation of stem cells. Our results also show the importance of considering the source and state of exosome donor cells before a choice is made for therapeutic applications.

A hyaluronan-based scaffold for the in vitro construction of dental pulp-like tissue.

Dental pulp tissue supports the vitality of the tooth, but it is particularly vulnerable to external insults, such as mechanical trauma, chemical irritation or microbial invasion, which can lead to tissue necrosis. In the present work, we present an endodontic regeneration method based on the use of a tridimensional (3D) hyaluronan scaffold and human dental pulp stem cells (DPSCs) to produce a functional dental pulp-like tissue in vitro. An enriched population of DPSCs was seeded onto hyaluronan-based non-woven meshes in the presence of differentiation factors to induce the commitment of stem cells to neuronal, glial, endothelial and osteogenic phenotypes. In vitro experiments, among which were gene expression profiling and immunofluorescence (IF) staining, proved the commitment of DPSCs to the main components of dental pulp tissue. In particular, the hyaluronan-DPSCs construct showed a dental pulp-like morphology consisting of several specialized cells growing inside the hyaluronan fibers. Furthermore, these constructs were implanted into rat calvarial critical-size defects. Histological analyses and gene expression profiling performed on hyaluronan-DPSCs grafts showed the regeneration of osteodentin-like tissue. Altogether, these data suggest the regenerative potential of the hyaluronan-DPSC engineered tissue.