Tooth Regeneration Research Articles

Odontogenesis-related developmental microenvironment facilitates deciduous dental pulp stem cell aggregates to revitalize an avulsed tooth

Harnessing developmental processes for tissue engineering represents a promising yet challenging approach to regenerative medicine. Tooth avulsion is among the most serious traumatic dental injuries, whereas functional tooth regeneration remains uncertain. Here, we established a strategy using decellularized tooth matrix (DTM) combined with human dental pulp stem cell (hDPSC) aggregates to simulate an odontogenesis-related developmental microenvironment. The bioengineered teeth reconstructed by this strategy regenerated three-dimensional pulp and periodontal tissues equipped with vasculature and innervation in a preclinical pig model after implantation into the alveolar bone. These results prompted us to enroll 15 patients with avulsed teeth after traumatic dental injuries in a pilot clinical trial. At 12 months after implantation, bioengineered teeth led to the regeneration of functional teeth, which supported continued root development, in humans. Mechanistically, exosomes derived from hDPSC aggregates mediated the tooth regeneration process by upregulating the odontogenic and angiogenic ability of hDPSCs. Our findings suggest that odontogenic microenvironment engineering by DTM and stem cell aggregates initiates functional tooth regeneration and serves as an effective treatment for tooth avulsion.

Spatiotemporal Changes in Transcriptome of Odontogenic and Non-odontogenic Regions in the Dental Arch of Mus musculus.

Over the past 40 years, studies on tooth regeneration have been conducted. These studies comprised two main flows: some focused on epithelial–mesenchymal interaction in the odontogenic region, whereas others focused on creating a supernumerary tooth in the non-odontogenic region. Recently, the scope of the research has moved from conventional gene modification and molecular therapy to genome and transcriptome sequencing analyses. However, these sequencing data have been produced only in the odontogenic region. We provide RNA-Seq data of not only the odontogenic region but also the non-odontogenic region, which loses tooth-forming capacity during development and remains a rudiment. Sequencing data were collected from mouse embryos at three different stages of tooth development. These data will expand our understanding of tooth development and will help in designing developmental and regenerative studies from a new perspective.

Induced Periapical Bleeding in Regeneration of Immature Permanent Teeth with Necrosis: A Systematic Review

Induced Periapical Bleeding in Regeneration of Immature Permanent Teeth with Necrosis: A Systematic Review

Wnt Signalling in Regenerative Dentistry

Teeth are complex structures where a soft dental pulp tissue is enriched with nerves, vasculature and connective tissue and encased by the cushioning effect of dentin and the protection of a hard enamel in the crown and cementum in the root. Injuries such as trauma or caries can jeopardise these layers of protection and result in pulp exposure, inflammation and infection. Provision of most suitable materials for tooth repair upon injury has been the motivation of dentistry for many decades. Wnt signalling, an evolutionarily conserved pathway, plays key roles during pre- and post-natal development of many organs including the tooth. Mutations in the components of this pathway gives rise to various types of developmental tooth anomalies. Wnt signalling is also fundamental in the response of odontoblasts to injury and repair processes. The complexity of tooth structure has resulted in diverse studies looking at specific compartments or cell types of this organ. This review looks at the current advances in the field of tooth development and regeneration. The objective of the present review is to provide an updated vision on dental biomaterials research, focusing on their biological properties and interactions to act as evidence for their potential use in vital pulp treatment procedures. We discuss the outstanding questions and future directions to make this knowledge more translatable to the clinics.

Increasing Odontoblast-like Differentiation from Dental Pulp Stem Cells through Increase of β-Catenin/p-GSK-3β Expression by Low-Frequency Electromagnetic Field.

Odontoblasts produce proteins that form the dentinal extracellular matrix, which can protect the dental pulp from external stimuli and is required for tooth regeneration. This study showed that a pulsed electromagnetic field (PEMF) can regulate cell metabolism and induce cell differentiation. This study determined the frequency of PEMF that is effective for odontoblast differentiation. Human dental pulp stem cells (hDPSCs) were cultured in odontoblast differentiation medium containing dexamethasone, BMP2, TGF-β1, and FGF-2, and then exposed to 10 mT intensity of PEMF at 40, 60, 70, and 150 Hz for 15 min/day. The MTT assay, LDH assay, flow cytometry, protein and gene expression, and immunofluorescence were performed to check if hDPSCs differentiated into odontoblast-like cells. The hDPSCs showed frequency-dependent differences in protein and gene expression. The mesenchymal stem cell markers were reduced to a greater extent at 60 and 70 Hz than at other frequencies, and odontoblast-related markers, particularly β-catenin, p-GSK-3β, and p-p38, were increased at 60 and 70 Hz. Exposure to 10 mT intensity of PEMF at 70 Hz influenced the differentiation of hDPSCs considerably. Taken together, PEMF treatment can promote differentiation of hDPSCs into odontoblast-like cells by increasing p-GSK-3β and β-catenin expression.

Exploitation Characteristics of Teeth Flanks of Gears Regenerated by Three Hard-Facing Procedures.

Numerous phenomena that occur during the process of machine parts’ regeneration have a significant impact on the loss of their working ability. Therefore, the properties of the working surfaces of the teeth flanks of repaired gears were analyzed in this research. The hereditary properties of the gear teeth are expressed by the interdependence of their geometric and physical-mechanical-metallurgical parameters created during the technological operations of regeneration of worn teeth by welding/hard-facing. The hard-facing was executed with three filler metal types, namely: combination Inox 18/8/6 + EDur 600, Castolin 2 and UTP 670. The tested properties included geometrical accuracy, microstructure and microhardness. Evaluation of the executed regeneration procedures was done by comparing the mentioned parameters of the regenerated gears and the new ones. The tested gears were not withdrawn from production due to damage, but they were newly manufactured and intentionally damaged gears, made of the same materials, subjected to the same manufacturing process. In this way, all influences except for the considered filler metal type were eliminated. Based on results of the conducted experiments, it was possible to establish the influence of the filler metal type on the surface characteristics of the regenerated gears’ teeth flanks.

Strategies for differentiation of hiPSCs into dental epithelial cell lineage

Different stem cell-based strategies, especially induced pluripotent stem cells (iPSCs), have been exploited to regenerate teeth or restore biological and physiological functions after tooth loss. Further research is needed to establish an optimized protocol to effectively differentiate human iPSCs (hiPSCs) into dental epithelial cells (DECs). In this study, various factors were precisely modulated to facilitate differentiation of hiPSCs into DECs, which are essential for the regeneration of functional teeth. Embryoid bodies (EBs) were formed from hiPSCs as embryo-like aggregates, retinoic acid (RA) was used as an early ectodermal inducer, and bone morphogenic protein 4 (BMP4) activity was manipulated. The characteristics of DECs were enhanced and preserved after culture in keratinocyte serum-free medium (K-SFM). The yielded cell population exhibited noticeable DEC characteristics, consistent with the expression of epithelial cell and ameloblast markers. DECs demonstrated odontogenic abilities by exerting an inductive effect on human dental pulp stem cells (hDPSCs) and forming a tooth-like structure with the mouse tooth mesenchyme. Overall, our differentiation protocol provides a practical approach for applying hiPSCs for tooth regeneration.

Identification of novel candidate genes implicated in odontogenic potential in the developing mouse tooth germ using transcriptome analysis.

In tooth bioengineering for replacement therapy of missing teeth, the utilized cells must possess an inductive signal-forming ability to initiate odontogenesis. This ability is called odontogenic potential. In mice, the odontogenic potential signal is known to be translocated from the epithelium to the mesenchyme at the early bud stage in the developing molar tooth germ. However, the identity of the molecular constituents of this process remains unclear. The purpose of this study is to determine the molecular identity of odontogenic potential and to provide a new perspective in the field of tooth development research. In this study, whole transcriptome profiles of the mouse molar tooth germ epithelium and mesenchyme were investigated using the RNA sequencing (RNA-seq) technique. The analyzed transcriptomes corresponded to two developmental stages, embryonic day 11.5 (E11.5) and 14.5 (E14.5), which represent the odontogenic potential shifts. We identified differentially expressed genes (DEGs), which were specifically overexpressed in both the E11.5 epithelium and E14.5 mesenchyme, but not expressed in their respective counterparts. Of the 55 DEGs identified, the top three most expressed transcription factor genes (transcription factor AP-2 beta isoform 3 [TFAP2B], developing brain homeobox protein 2 [DBX2], and insulin gene enhancer protein ISL-1 [ISL1]) and three tooth development-related genes (transcription factor HES-5 [HES5], platelet-derived growth factor D precursor [PDGFD], semaphrin-3A precursor [SEMA3A]) were selected and validated by quantitative RT-PCR. Using immunofluorescence staining, the TFAP2B protein expression was found to be localized only at the E11.5 epithelium and E14.5 mesenchyme. Thus, our empirical findings in the present study may provide a new perspective into the characterization of the molecules responsible for the odontogenic potential and may have an implication in the cell-based whole tooth regeneration strategy.

Current natural bioactive materials in bone and tooth regeneration in dentistry: a comprehensive overview

Tissue regeneration is a vital phenomenon in the skeletal system of human beings during their life span. The structure of bony architecture and teeth are highly dependent to this ongoing procedure to maintain their functional form. Regarding the fundamental function of our skeletal system, all the involved components are at constant risk for injuries and deformations throughout the life. Therefore, in modern medicine, promotion of bone and tooth regeneration has become a critical counter measure to correct these acquired defects. There are numerous ways to address the bone and tooth related erosive pathologies but utilization of natural compounds has gained immense popularity in recent years. These materials provide a broad spectrum of therapeutic agents with a variety of biological characteristics. In this review, a comprehensive exploration has been implemented to extract and categorize the relevant scientific evidence on this issue, based on the source of natural candidates and the potential clinical indications. Database searching presented a considerable data pool with more than 300 references in this field. According to the results, all the proposed items can be categorized in two major groups including scaffolding and osteogenesis (or dentinogenesis) induction. The clinical applicability of a number of them is properly demonstrated in clinical trials but for the rest, more examinations are required to reach a conclusive decision.

The ability of platelet-rich plasma to regenerate a non-vital immature permanent teeth

Objectives: To test the ability of platelet-rich plasma clinically and radiologically for pulp regeneration of immature teeth with apical periodontitis. Material and Methods: An experimental study was conducted From (March/2018-July/2020) 12 upper central immature incisors with acute apical periodontitis and necrotic pulp from six patients receiving regenerative endodontic treatment using concentrated platelets rich plasma were performed by the same endodontist at Mediclinic Middle East Hospitals. Informed consent, including explanation of risks and alternative treatments or no treatment were prepared and filled by the patient parents. The therapeutic protocol was involved accessing the pulp chamber; irrigation copiously with sodium hypochlorite; applying calcium hydroxide as intracanal medicament and a provisionally sealing it after 4 weeks. The canal was cleaned, dried and injected with concentrated platelets rich plasma which serve as a scaffold for pulp regeneration. MTA was used to seal the chamber before final filling with composite. Evaluations: All teeth were monitored clinically (mobility, palpation, percussion, and sensitivity cold test) and radiographically. Results: Twenty months follow-up all teeth showed resolution of periapical radiolucencies, continued root development with positive response to sensitivity cold test and no discoloration. Conclusion: The results of this study confirmed the previous finding that pulp regeneration can be gained by using cPRP successfully Keywords Immature teeth; Necrosis; cPRP; Regeneration.

Prospective cohort study of regenerative potential of non vital immature permanent maxillary central incisors using platelet rich fibrin scaffold

Regenerative endodontic procedures have gained momentum as a treatment modality of young immature permanent teeth. Literature reports reveal that platelet-rich fibrin (PRF) stimulates growth factors and induces regeneration. This study was undertaken to assess the regenerative potential of non-vital immature permanent maxillary central incisors using PRF with a follow-up for 2 yrs. 19 patients in the age group of 9–25 yrs with immature, non-vital permanent maxillary central incisors (n = 23) with/without signs and/or symptoms of periapical pathosis and open apex were included in this study. In the first appointment, access opening, canal disinfection and triple antibiotic paste placement were done. In the subsequent visit, PRF was prepared and placed inside the canal. Access was sealed with Mineral trioxide aggregate plug and composite. The patient was reviewed up to 24 mths. The mean difference was statistically analyzed using Friedman test followed by Dunn post hoc test and adjusted by Bonferroni correction (p < 0.05). As per AAE guidelines, the primary and secondary goals were achieved. A significant (p < 0.001) gradual increase in the root length, thickness of dentinal walls and decrease in apical diameter were observed. Within the limitations of this study, PRF placement was clinically and radiographically effective in inducing regeneration of non-vital immature permanent teeth.

Local application of Usag-1 siRNA can promote tooth regeneration in Runx2-deficient mice

Runt-related transcription factor 2 (Runx2)-deficient mice can be used to model congenital tooth agenesis in humans. Conversely, uterine sensitization-associated gene-1 (Usag-1)-deficient mice exhibit supernumerary tooth formation. Arrested tooth formation can be restored by crossing both knockout-mouse strains; however, it remains unclear whether topical inhibition of Usag-1 expression can enable the recovery of tooth formation in Runx2-deficient mice. Here, we tested whether inhibiting the topical expression of Usag-1 can reverse arrested tooth formation after Runx2 abrogation. The results showed that local application of Usag-1 Stealth small interfering RNA (siRNA) promoted tooth development following Runx2 siRNA-induced agenesis. Additionally, renal capsule transplantation of siRNA-loaded cationized, gelatin-treated mouse mandibles confirmed that cationized gelatin can serve as an effective drug-delivery system. We then performed renal capsule transplantation of wild-type and Runx2-knockout (KO) mouse mandibles, treated with Usag-1 siRNA, revealing that hindered tooth formation was rescued by Usag-1 knockdown. Furthermore, topically applied Usag-1 siRNA partially rescued arrested tooth development in Runx2-KO mice, demonstrating its potential for regenerating teeth in Runx2-deficient mice. Our findings have implications for developing topical treatments for congenital tooth agenesis.

Importance of In Vitro Microenvironment on Differentiation and Translational Applications of Dental Pulp Stem Cells

Dental Pulp Stem Cells (DPSCs) are adult stem cells found in dental pulp tissue, and possess the capacity for self-renewal and the potential for multipotent differentiation. DPSCs depend on regulating in vitro microenvironment, and can readily differentiate into osteoblasts, odontoblasts, neurocytes, adipocytes, chondrocytes, myocytes, fibrocytes and many others. In addition, DPSCs play a crucial role in tooth regeneration, bone and nerve repair in current studies of regenerative medicine and tissue engineering. Among them, the influences of stem cell microenvironment or niche on the biological activity of DPSCs are critical and hamper its progress. Herein, we review the influence of culture condition, tissue source, growth factor requirements, and cellular organizational scaffolds and how these features influence the biological characteristics and translational research of DPSCs.

Characteristics of Large Animal Models for Current Cell-Based Oral Tissue Regeneration.

The recent advances in the field of cell-based therapeutics open promising perspectives for oral tissue regeneration. The development of large animal models, which overcome the limits of the rodent models and allow to emulate clinical situations, is crucial for the validation of regenerative strategies to move toward clinical application. Currently, porcine, canine, and ovine models are mainly developed for oral regeneration and their specific characteristics have an impact on the outcomes of the studies. Thus, this systematic review investigates the application of porcine, canine, and ovine models in present cell-based oral regeneration, according to the species characteristics and the targeted tissue to regenerate. A customized search of PubMed, EMBASE, Scopus, and Web of Science databases from January 2015 to March 2020 was conducted. Relevant articles about cell-based oral tissues engineering in porcine, canine, and ovine models were evaluated. Among the evaluated articles, 58 relevant studies about cell-based oral regeneration in porcine, canine, and ovine models matched the eligibility criteria and were selected for full analysis. Porcine models, the most similar species with humans, were mostly used for bone and periodontium regeneration; tooth regeneration was reported only in pig, except for one study in dog. Canine models were the most transversal models, successfully involved for all oral tissue regeneration and notably in implantology. However, differences with humans and ethical concerns affect the use of these models. Ovine models, alternative to porcine and canine ones, were mainly used for bone and, scarcely, periodontium regeneration. The anatomy and physiology of these animals restrain their involvement. If consistency was found in defect specificities and cell trends among different species animal models of bone, dentin-pulp complex, or tooth regeneration, variability appeared in periodontium. Regeneration assessment methods were more elaborate in porcines and canines than in ovines. Risk of bias was low for selection, attrition and reporting, but unclear for performance and detection. Overall, if none of the large animal models can be considered an ideal one, they are of deemed importance for oral cell-based tissue engineering and researchers should consider their relevance to establish favorable conditions for a given preclinical cell-based therapeutics. Impact statement This systematic review investigates porcine, canine, and ovine models for current oral cell-based regeneration procedures, and researchers could refer to it for the choice of the most pertinent preclinical model for a given cell-based therapeutics.

Treated dentin matrix induces odontogenic differentiation of dental pulp stem cells via regulation of Wnt/β-catenin signaling

Treated dentin matrix (TDM) is an ideal scaffold material containing multiple extracellular matrix factors. The canonical Wnt signaling pathway is necessary for tooth regeneration. Thus, this study investigated whether the TDM can promote the odontogenic differentiation of human dental pulp stem cells (hDPSCs) and determined the potential role of Wnt/β-catenin signaling in this process. Different concentrations of TDM promoted the dental differentiation of the hDPSCs and meanwhile, the expression of GSK3β was decreased. Of note, the expression of the Wnt/β-catenin pathway-related genes changed significantly in the context of TDM induction, as per RNA sequencing (RNA seq) data. In addition, the experiment showed that new dentin was visible in rat mandible cultured with TDM, and the thickness was significantly thicker than that of the control group. In addition, immunohistochemical staining showed lower GSK3β expression in new dentin. Consistently, the GSK3β knockdown hDPSCs performed enhanced odotogenesis compared with the control groups. However, GSK3β overexpressing could decrease odotogenesis of TDM-induced hDPSCs. These results were confirmed in immunodeficient mice and Wistar rats. These suggest that TDM promotes odontogenic differentiation of hDPSCs by directly targeting GSK3β and activating the canonical Wnt/β-catenin signaling pathway and provide a theoretical basis for tooth regeneration engineering.

Multidifferentiation potential of dental-derived stem cells.

Tooth-related diseases and tooth loss are widespread and are a major public health issue. The loss of teeth can affect chewing, speech, appearance and even psychology. Therefore, the science of tooth regeneration has emerged, and attention has focused on tooth regeneration based on the principles of tooth development and stem cells combined with tissue engineering technology. As undifferentiated stem cells in normal tooth tissues, dental mesenchymal stem cells (DMSCs), which are a desirable source of autologous stem cells, play a significant role in tooth regeneration. Researchers hope to reconstruct the complete tooth tissues with normal functions and vascularization by utilizing the odontogenic differentiation potential of DMSCs. Moreover, DMSCs also have the ability to differentiate towards cells of other tissue types due to their multipotency. This review focuses on the multipotential capacity of DMSCs to differentiate into various tissues, such as bone, cartilage, tendon, vessels, neural tissues, muscle-like tissues, hepatic-like tissues, eye tissues and glands and the influence of various regulatory factors, such as non-coding RNAs, signaling pathways, inflammation, aging and exosomes, on the odontogenic/osteogenic differentiation of DMSCs in tooth regeneration. The application of DMSCs in regenerative medicine and tissue engineering will be improved if the differentiation characteristics of DMSCs can be fully utilized, and the factors that regulate their differentiation can be well controlled.

Multipotent stem cells from apical pulp of human deciduous teeth with immature apex

Isolation of high-quality human postnatal stem cells from accessible sources is an important goal for dental tissue engineering. Stem cells from developing organs are a better cell source but are hard to obtain. With extensive caries that are difficult to restore, the extracted deciduous tooth with an immature apex is a developing organ for investigation. In the present study, a cell population from the tip of apical pulp of human deciduous teeth with an immature apex was isolated and termed apical pulp-derived cells of deciduous teeth (De-APDCs). De-APDCs expressed STRO-1, CD44, CD90 and CD105 but not CD34 or CD45. Furthermore, De-APDCs demonstrated a significantly higher clonogenic and proliferative ability and osteo/dentinogenic differentiation capacity than dental pulp cells from exfoliated deciduous teeth (De-DPCs) (P < 0.05). Differentiation potential toward adipogenic, neurogenic and chondrogenic lineages was also observed in induced De-APDCs. In addition, after De-APDCs were seeded into hydroxyapatite/tricalcium phosphate (HA/TCP) scaffolds and transplanted into nude mice, they were able to regenerate dentin/pulp-like structures aligned with human odontoblast-like cells. In conclusion, De-APDCs, which are derived from a developing tissue, represent an accessible and prospective cell source for tooth regeneration.

Eruption of Bioengineered Teeth: A New Approach Based on a Polycaprolactone Biomembrane.

Obtaining a functional tooth is the ultimate goal of tooth engineering. However, the implantation of bioengineered teeth in the jawbone of adult animals never allows for spontaneous eruption due mainly to ankylosis within the bone crypt. The objective of this study was to develop an innovative approach allowing eruption of implanted bioengineered teeth through the isolation of the germ from the bone crypt using a polycaprolactone membrane (PCL). The germs of the first lower molars were harvested on the 14th day of embryonic development, cultured in vitro, and then implanted in the recipient site drilled in the maxillary bone of adult mice. To prevent the ankylosis of the dental germ, a PCL membrane synthesized by electrospinning was placed between the germ and the bone. After 10 weeks of follow-up, microtomography, and histology of the implantation site were performed. In control mice where germs were directly placed in contact with the bone, a spontaneous eruption of bioengineered teeth was only observed in 3.3% of the cases versus 19.2% in the test group where PCL biomembrane was used as a barrier (p < 0.1). This preliminary study is the first to describe an innovative method allowing the eruption of bioengineered tooth implanted directly in the jawbone of mice. This new approach is a hope for the field of tooth regeneration, especially in children with oligodontia in whom titanium implants are not an optimal solution.

Tooth Removal in the Leopard Gecko and the de Novo Formation of Replacement Teeth

Introduction and Objectives: The ability to replace teeth throughout life is a trait found in most vertebrates and was present in ancient mammals. Modern mammals including humans can only replace their teeth once and much effort is currently being devoted to uncovering stem cells to be used in tooth regeneration. Work from our lab and others has identified some of the differences in the reptilian dentition that could underlie the basis of polyphyodonty. In lizards, the dental lamina, a source of putative dental epithelial progenitor cells, persists until adulthood. In contrast, in humans there are very few remnants of the dental lamina remaining once the permanent dentition is complete. In this project we use the leopard gecko (Eublepharis macularius) and surgical treatments to trace the contribution of label-retaining cells to the next generation of teeth. We hypothesized that premature removal of unerupted second generation teeth will stimulate populations of quiescent label-retaining cells to proliferate. Then by following the labeled cells for several months we will identify progeny of the epithelial progenitor cells in newly formed teeth. Materials and Methods: Second generation, unerupted teeth were removed from geckos under sedation. A pulse of BrdU was administered over 3 sequential days. Animals were euthanized at 1 week post surgery (immediately after the BrdU pulse or time 0) and 1 or 2 months post surgery (N = 3 animals per timepoint). Animals were processed for immunofluorescence staining with BrdU antibodies. We counted the proportion of nuclei in the dental lamina that stained positive for BrdU. Results: One week after the surgery, we identified statistically significant higher uptake of BrdU in the dental epithelium of treated as compared to the control, non-manipulated regions (P < 0.01). One month after the surgery, there was a significant decrease in BrdU label in the dental lamina (P< 0.001), decreasing to the same level as controls at time zero The newly formed tooth buds regenerating in the sites of tooth removal contained BrdU labeled cells in all parts of the enamel organ. The original label-retaining cells divided several times since the original pulse, based on the fragmented appearance of the BrdU in the nuclei. Conclusion: In our previous work, it was difficult to label the quiescent cells of the dental lamina. Here the removal of the successional teeth stimulates proliferation of the dental lamina. With a larger population of label-retaining cells that could be tracked we were able to find transit amplifying cells that divide and contribute to the enamel organ. Significance: We have developed an injury model that will allow us to test the ability of different signals to regulate the activity of stem cells. Moreover label-retaining cells are multipotent and can self-renew, thereby possessing some of the properties of progenitor cells.

Adipose Tissue–derived Microvascular Fragments as Vascularization Units for Dental Pulp Regeneration

IntroductionThe transplantation of dental pulp stem cells (DPSCs) has emerged as a novel strategy for the regeneration of lost dental pulp after pulpitis and trauma. Dental pulp regeneration of the young permanent tooth with a wide tooth apical foramen has achieved significant progress in the clinical trials. However, because of the narrow apical foramen, dental pulp regeneration in adult teeth using stem cells remains difficult in the clinic. Finding out how to promote vascular reconstitution is essential for the survival of stem cells and the regeneration of dental pulp after transplantation into the adult tooth. MethodsAdipose tissue–derived microvascular fragments (ad-MVFs) were isolated from human adipose tissues. The apoptosis and senescence of DPSCs cultured in conditioned media were evaluated to explore the effects of ad-MVFs on DPSCs. DPSCs combined with ad-MVFs were inserted into the human tooth root segments and implanted subcutaneously into immunodeficient mice. Regenerated pulplike tissues were analyzed by hematoxylin and eosin and immunohistochemistry. The vessels in regenerated tissues were analyzed by Micro-CT and immunofluorescence. ResultsThe isolated ad-MVFs contained endothelial cells and pericytes. ad-MVFs effectively prevented the apoptosis and senescence of the transplanted DPSCs both in vivo and in vitro. Combined with DPSCs, ad-MVFs obviously facilitated the formation of vascular networks in the transplants. DPSCs combined with ad-MVFs formed dental pulp–like tissues with abundant cells and matrix after 4 weeks of implantation. The supplementation of ad-MVFs led to more odontoblastlike cells and increased the formation of mineralized substance around the root canal. ConclusionsCotransplantation with ad-MVFs promotes the angiogenesis and revascularization of transplanted DPSC aggregates, leading to robust regeneration of dental pulp.