Proliferation of mature ex vivo human dental pulp using tissue engineering scaffolds

Introduction

The purpose of this study was to measure and compare the proliferation of mature human dental pulp tissue using three types of tissue engineering scaffolds.

Methods

Mature human teeth were collected immediately after extraction for routine dental treatment reasons. Three types of tissue engineering scaffolds were investigated (1) open-polylactic acid (polymer) scaffolds, (2) bovine collagen (collagen) scaffolds, and (3) calcium phosphate bioceramic (calcium phosphate) scaffolds. The scaffolds were placed in direct contact with the dental pulp of the tooth slices from 7 to 30 days. Neutral-red dye was added to the culture media to stain metabolically active cells. The specimens were processed for histology. The numbers of proliferating cells were counted per unit area of scaffold according to ISO criteria.

Results

The proliferating dental pulp cells had a fibroblast phenotype, no cells of other phenotypes were observed, and none of the cells appeared to be mineralizing. The average rate of mature vital dental cell proliferation was 1.305 cells per day in the calcium phosphate scaffolds compared with 7.195 (a rate increase of 551%) in the collagen scaffolds and 13.885 (a rate increase of 1,064%) in the polymer scaffolds.

Conclusions

Tissue engineering scaffolds can enhance the proliferation of mature dental pulp tissue. The rate of dental pulp proliferation is dependent on the chemical composition of the scaffold. Within the limitations of this study, the polymer scaffolds were more optimal than collagen or calcium phosphate scaffolds for mature dental pulp proliferation.     

成人牙髓干细胞与壳聚糖-磷酸三钙复合材料相容性试验研究

目的:研究成人牙髓干细胞与壳聚糖-磷酸三钙复合材料的生物相容性。方法:采用冷冻干燥法制备壳聚糖-磷酸三钙复合材料。酶消化法分离,培养人牙髓干细胞,并将达到一定数量级的牙髓干细胞与支架材料进行复合培养,通过扫描电镜观察细胞的生长情况。结果:扫描电镜可见复合材料具有良好的多孔网状结构,成人牙髓干细胞与材料表面紧密贴附,生长良好。结论:复合壳聚糖-磷酸三钙进行培养的成人牙髓干细胞生长良好,具有良好的生物相容性。表明壳聚糖-磷酸三钙完全符合生物支架材料的要求,是一个具有很好应用前景的牙髓组织工程支架材料。     

Evaluation of the Apical Complex and the Coronal Pulp as a Stem Cell Source for Dentin-pulp Regeneration

IntroductionThis study compared the stemness and differentiation potential of stem cells derived from the apical complex (apical complex cells [ACCs]) and coronal pulp (dental pulp stem cells [DPSCs]) of human immature permanent teeth with the aim of determining a more suitable source of stem cells for regeneration of the dentin-pulp complex.MethodsACC and DPSC cultures were established from 13 human immature permanent teeth using the outgrowth method. The proliferation capacity and colony-forming ability of ACCs and DPSCs were evaluated. ACCs and DPSCs were analyzed for mesenchymal stem cell markers using flow cytometry. The adipogenic and osteogenic differentiation potential of ACCs and DPSCs were evaluated using the quantitative real-time polymerase chain reaction and histochemical staining. ACCs and DPSCs were transplanted subcutaneously in immunocompromised mice using macroporous biphasic calcium phosphate as a carrier. The histomorphologic characteristics of the newly formed tissues were verified using hematoxylin-eosin staining and immunohistochemical staining. Quantitative alkaline phosphatase analysis and quantitative real-time polymerase chain reaction using BSP, DSPP, POSTN, and Col XII were performed.ResultsACCs and DPSCs showed similar cell proliferation potential and colony-forming ability. The percentage of mesenchymal stem cell markers was similar between ACCs and DPSCs. In the in vitro study, ACCs and DPSCs showed adipogenic and osteogenic differentiation potential. In the in vivo study, ACCs and DPSCs formed amorphous hard tissue using macroporous biphasic calcium phosphate particles. The quantity and histomorphologic characteristics of the amorphous hard tissue were similar in the ACC and DPSC groups. Formation of periodontal ligament–like tissue, positive to Col XII, was observed in ACC transplants, which was absent in DPSC transplants.ConclusionsACCs and DPSCs showed similar stemness, proliferation rate, and hard tissue–forming capacity. The notable difference was the periodontal ligament–like fiber-forming capacity of ACCs, which indicates the presence of various lineages of stem cells in the apical complex compared with the coronal pulp. Regarding regeneration of the dentin-pulp complex, the coronal pulp can be a suitable source of stem cells considering its homogenous lineages of cells and favorable osteo/odontogenic differentiation potential.     

凝胶样支架材料对三维培养的人牙髓干细胞增殖的影响

目的　评价牙髓组织工程中不同浓度的两种可注射性凝胶样支架材料对牙髓干细胞增殖的影响。方法：配制不同浓度的I型胶原、肽段水凝胶（Puramatrix）支架材料,将牙髓干细胞分别接种于各组支架材料上;CCK-8方法检测细胞增殖情况;活死细胞染色观察细胞数量及形态。 结果　接种在各浓度支架材料上的牙髓干细胞均生长良好,其中1%的胶原支架和0.25%的水凝胶支架对牙髓干细胞的增殖有明显的促进作用;活死细胞染色观察牙髓干细胞在各浓度的两种支架材料中均生长良好,细胞数量随培养时间的延长呈递增趋势;细胞充分伸展,呈纺锤型。     

Stand der humanen dentalen Stammzellforschung

This review article arranges the current results of stem cell biology for their use in dentistry. There are different types of stem cells, which are applicable for dental treatments. The use of embryonic stem cells, whose possibilities for breeding an artificial tooth were hardly evaluated, is however ethically precarious. On the other side the ethically harmless adult stem cells, which were isolated for example from bone marrow, were little examined for their capability of differentiation into dental tissues. Therefore their forthcoming use in dentistry is rather improbable. However, dental ectomesenchymal stem cells are more promising for dentistry in future. For example dental pulp stem cells (DPSCs) are capable to differentiate into dentin under in vitro conditions. Moreover it is possible to use periodontal ligament (PDL) stem cells and dental follicle precursors for periodontal tissue differentiations in vitro. Recently new populations of stem cells were isolated from the dental pulp and the PDL. These cells distinguish from the initially isolated DPSCs and PDL stem cells in growth and cell differentiation. Therefore stem cell markers are very important for the characterization of dental stem cells. A significant marker for dental stem cells is STRO-1, which is also a marker for bone marrow derived mesenchymal stem cells. Nonetheless dental stem cells are CD45 negative and they express rarely hematopoietic stem cell markers. These research results plead for the participation of dental stem cells in dental practice in future.     

牙髓干细胞分化过程中L型钙离子通道羧基末端的表达

目的:探讨牙髓干细胞(DPSCs)分化过程中L型钙离子通道羧基末端的表达。方法:利用酶消化法体外分离、培养大鼠牙髓干细胞;吉姆萨染色法检测大鼠牙髓干细胞的克隆形成能力;神经诱导体系下诱导牙髓干细胞向神经样细胞分化,免疫荧光染色检测细胞分化后胶质纤维酸蛋白(glial fibrillary acidic pro-tein,GFAP)的表达和细胞分化前后L型钙离子通道Cav 1.2及羧基末端的表达。结果:牙髓干细胞的克隆形成能力为每1 000个细胞形成2～17个克隆;免疫荧光染色检测诱导后细胞GFAP表达阳性;免疫荧光染色检测显示:牙髓干细胞分化前L型钙离子通道Cav 1.2羧基末端表达于细胞膜上,细胞分化后羧基末端同时表达于细胞膜上和细胞核中。结论:L型钙离子通道Cav 1.2羧基末端在牙髓干细胞分化过程中发生核转位,羧基末端可能在牙髓干细胞的分化过程中发挥着一定的作用。     

Preliminary Study on Dental Pulp Stem Cell–mediated Pulp Regeneration in Canine Immature Permanent Teeth

IntroductionThe health of human teeth depends on the integrity of the hard tissue and the activity of the pulp and periodontal tissues, which are responsible for nutritional supply. Without the nourishing of the pulp tissue, the possibility of tooth fracture can increase. In immature permanent teeth, root development may be influenced as well. This study explored the potential of using autologous dental pulp stem cells (DPSCs) to achieve pulp regeneration in a canine pulpless model.MethodsThe establishment of the pulpless animal model involved pulp extirpation and root canal preparation of young permanent incisor teeth in beagles. Autologous DPSCs were obtained from extracted first molars and expanded ex vivo to obtain a larger number of cells. The biological characteristics of canine DPSCs (cDPSCs) were analyzed both in vitro and in vivo by using the same method as used in human DPSCs. cDPSCs were transplanted into the pulpless root canal with Gelfoam as the scaffold, and root development was evaluated by radiographic and histologic analyses.ResultscDPSCs with rapid proliferation, multiple differentiation capacity, and development potential were successfully isolated and identified both in vitro and in vivo. After they were transplanted into the pulpless root canal with Gelfoam as the scaffold, DPSCs were capable of generating pulp-like tissues containing blood vessels and dentin-like tissue. Thickening of the root canal wall was also observed.ConclusionsThis study demonstrates the feasibility of using stem cell–mediated tissue engineering to realize pulp regeneration in immature teeth.     

Biomimetic Approach to Perforation Repair Using Dental Pulp Stem Cells and Dentin Matrix Protein 1

Introduction

Dentin regeneration could be an ideal treatment option to restore tissue function. This study was conducted to evaluate the ability of dental pulp stem cells (DPSCs) and dentin matrix protein 1 (DMP1) impregnated within a collagen scaffold to regenerate dentin.

Methods

Simulated perforations were created in 18 dentin wafers made from freshly extracted human molars. Six groups were established. They were (1) empty wafers, (2) mineral trioxide aggregate, (3) collagen scaffold, (4) scaffold with DMP1, (5) scaffold with DPSCs, and (6) scaffold with DPSCs and DMP1. One sample was placed subcutaneously in each mouse with three mice in each group. After 12 weeks, the samples were subjected to radiographic, histological, and immunohistochemical evaluations.

Results

DPSCs impregnated within a collagen scaffold differentiated into odontoblast-like cells forming a highly cellular, vascular, and mineralized matrix in the presence of DMP1.

Conclusions

A triad consisting of DPSCs, DMP1, and a collagen scaffold promotes dentin regeneration in a simulated perforation repair model.     

牙髓干细胞在口腔颌面部骨组织工程中的应用进展

口腔头面部疾病及外伤均可导致口腔颌面部及牙齿周围骨组织缺损。组织工程骨是修复口腔颌面部骨和牙周组织缺损的重要手段,而理想的种子细胞是得到组织工程骨的必要条件。牙髓干细胞容易获取,病原携带率低,培养效率高,具有成骨分化能力,与多种生物活性材料亲和性高,是组织工程修复骨缺损的理想种子细胞。本文对牙髓干细胞在口腔颌面部及牙周组织工程骨中的应用研究进展进行综述。     

Stem cell‐based tooth and periodontal regeneration

Currently regeneration of tooth and periodontal damage still remains great challenge. Stem cell‐based tissue engineering raised novel therapeutic strategies for tooth and periodontal repair. Stem cells for tooth and periodontal regeneration include dental pulp stem cells (DPSCs), periodontal ligament stem cells (PDLSCs), stem cells from the dental apical papilla (SCAPs), and stem cells from human exfoliated deciduous teeth (SHEDs), dental follicle stem cells (DFSCs), dental epithelial stem cells (DESCs), bone marrow mesenchymal stem cells (BMMSCs), adipose‐derived stem cells (ADSCs), embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs). To date, substantial advances have been made in stem cell‐based tooth and periodontal regeneration, including dentin–pulp, whole tooth, bioroot and periodontal regeneration. Translational investigations have been performed such as dental stem cell banking and clinical trials. In this review, we present strategies for stem cell‐based tissue engineering for tooth and periodontal repair, and the translational studies.     

人牙髓干细胞的体外培养和鉴定

目的　研究第三恒磨牙来源的人牙髓干细胞的表型和生物学性状。方法　从成人健康阻生牙中获取牙髓,酶消化法分离获得牙髓干细胞,计算细胞克隆形成率(CFU-F);免疫组化、RT-PCR法检测细胞的表面分子表达; 流式细胞仪测定细胞周期;体外分化诱导实验检测细胞的多向分化能力。结果　分离获得的牙髓干细胞在体外具有一定的克隆形成能力,诱导条件下部分牙髓干细胞可向脂肪、肌细胞和成牙本质细胞方向分化,符合干细胞的特征。结论　成功的从人第三恒磨牙牙髓中分离得到牙髓干细胞。     

Effect of leptin on differentiation of human dental stem cells

Oral Diseases (2011) 17 , 662–669 Objectives: Mesenchymal stem cells (MSCs) were identified in adult human periodontal ligament and dental pulp that are considered as potential stem cell sources for future clinical applications in dentistry. Leptin is known as an important regulator of mesenchymal differentiation. The objective of this study was to elucidate the role of leptin on proliferation and differentiation of dental MSCs. Materials and methods: Enhancement of cemento/odontoblastic differentiation of dental stem cells by leptin was confirmed by alizarin red S staining and alkaline phosphatase activity staining. In contrast, leptin reduced adipogenesis in both dental pulp stem cells (DPSCs) and periodontal ligament stem cells (PDLSCs) confirmed by oil red O staining and RT‐PCR. The expression of adipogenic markers, lipoprotein lipase and proliferator‐activated receptor γ2 (PPARγ2), were suppressed in PDLSCs incubated on media supplemented with leptin for 2 weeks. Results: Leptin had a relatively stronger osteogenesis promoting effect and adipogenesis suppressing effect in PDLSCs than in DPSCs. Conclusions: Collectively, leptin had a relatively stronger promoting effect on cemento/odontoblastic differentiation and a suppressing effect on adipogenesis in PDLSCs than in DPSCs. This study has provided evidence that leptin acts as an important modulator of dental MSCs differentiation.     

Dental pulp stem cells in chitosan/gelatin scaffolds for enhanced orofacial bone regeneration

Objective

Biomimetic chitosan/gelatin (CS/Gel) scaffolds have attracted great interest in tissue engineering of several tissues. However, limited information exists regarding the potential of combining CS/Gel scaffolds with oral cells, such as dental pulp stem cells (DPSCs), to produce customized constructs targeting alveolar/orofacial bone reconstruction, which has been the aim of the present study.

Enhancing cell seeding and osteogenesis of MSCs on 3D printed scaffolds through injectable BMP2 immobilized ECM-Mimetic gel

ObjectiveDesign of bioactive scaffolds with osteogenic capacity is a central challenge in cell-based patient-specific bone tissue engineering. Efficient and spatially uniform seeding of (stem) cells onto such constructs is vital to attain functional tissues. Herein we developed heparin functionalized collagen gels supported by 3D printed bioceramic scaffolds, as bone extracellular matrix (ECM)-mimetic matrices. These matrices were designed to enhance cell seeding efficiency of mesenchymal stem cells (MSCs) as well as improve their osteogenic differentiation through immobilized bone morphogenic protein 2 (BMP2) to be used for personalized bone regeneration.MethodsA 3D gel based on heparin-conjugated collagen matrix capable of immobilizing recombinant human bone morphogenic protein 2 (BMP2) was synthesized. Isolated dental pulp Mesenchymal stem cells (MSCs) were then encapsulated into the bone ECM microenvironment to efficiently and uniformly seed a bioactive ceramic-based scaffold fabricated using additive manufacturing technique. The designed 3D cell-laden constructs were comprehensively investigated trough in vitro assays and in vivo study.ResultsIn-depth rheological characterizations of heparin-conjugated collagen gel revealed that elasticity of the matrix is significantly improved compared with freely incorporated heparin. Investigation of the MSCs laden collagen-heparin hydrogels revealed their capability to provide spatiotemporal bioavailability of BMP2 while suppressing the matrix contraction over time. The in vivo histology and real-time polymerase chain reaction (qPCR) analysis showed that the designed construct supported the osteogenic differentiation of MSCs and induced the ectopic bone formation in rat model.SignificanceThe presented hybrid constructs combine bone ECM chemical cues with mechanical function providing an ideal 3D microenvironment for patient-specific bone tissue engineering and cell therapy applications. The implemented methodology in design of ECM-mimetic 3D matrix capable of immobilizing BMP2 to improve seeding efficiency of customized scaffolds can be exploited for other bioactive molecules.     

Cell-derived Extracellular Matrix Proteins in Colloidal Microgel as a Self-Assembly Hydrogel for Regenerative Endodontics

IntroductionThis study investigated a colloidal microgel for angiogenic and odontogenic differentiation of cells in the presence of cell-derived extracellular matrix (ECM) proteins using a 3-dimensional culture model.MethodsViscoelastic properties of human dental pulp were determined to understand the native ECM environment. ECM proteins were extracted from dental pulp stem cell (DPSC) cultures, and MaxGel (Millipore Sigma, Burlington, MA) was used as a commercially available ECM protein. DPSCs were incubated in colloidal microgels in the presence of ECM proteins or gelatin methacryloyl (GelMA) as a bulk hydrogel (n = 9/group). The viability and odontogenic differentiation of DPSCs within hydrogels was determined using viability assays, mineralization staining, calcium and alkaline phosphatase assays, and quantitative polymerase chain reaction for odontogenic gene expression. Angiogenic properties of endothelial cells were determined using tubule formation assays and quantitative polymerase chain reaction to detect angiogenic gene expression.ResultsDental pulp had a higher elastic modulus than the viscous modulus, showing a solidlike response similar to hydrogels. DPSC-derived ECM showed higher collagen and GAG than MaxGel (P < .05). The viability of DPSCs was similar in colloidal microgels, whereas higher cell viability, calcium deposition, and alkaline phosphatase activity were observed in GelMA (P < .05). Colloidal microgels allowed tubule-like structures by endothelial cells, whereas no tubular formation was observed in GelMA. DPSC-derived ECM in colloidal microgel up-regulated odontogenic gene expression, whereas MaxGel up-regulated angiogenic gene expression (P < .05).ConclusionsColloidal microgels allowed cellular organization that can improve penetration and nutritional supply in a full-length root canal system. The bioactivity of cell-derived ECM proteins can be modified depending on the external stimulus.     

Dental stem cells: Progress and perspectives

Dental pulp stem cells (DPSCs) are thought to contribute to reparative dentin formation, and that they may correspond to heterogenous populations of precursor cells or represent distinct differentiation stages along the odontoblastic lineage. DPSCs share many similarities with mesenchymal stem cells of the bone marrow (BMSCs). It appears that the distribution of tissue stem cells is not random and, within the dental pulp, there are potentially several distinct niches of stem/progenitor cells. In addition to DPSCs, other dental stem cell populations have been isolated. As for DPSCs, further studies are still needed to evaluate their potential of differentiation and their regenerative activity. Up today, (1) the formal demonstration that pulpal resident stem cells are actually the reparative dentin-forming cells recruited in response to injury is still lacking; and (2) the origin, localization and precise identity of odontogenic stem cells remain largely unknown. Dental clonal cell lines may represent valuable tool to answer some fontamental questions concerning the dental stem cell biology. Altogether, the presence of dental cell populations displaying stem cell properties has opened new paths for considering regenerative therapies. This might be a prerequisite to design alternative strategies for capping and endodontic treatment, using stem 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.     

Mesenchymal Stem Cells Derived from Human Inflamed Dental Pulp Exhibit Impaired Immunomodulatory Capacity In Vitro

IntroductionDental pulp stem cells (DPSC) are very attractive in regenerative medicine. In this study, we focused on the characterization of the functional properties of mesenchymal stem cells derived from DPSCs. Currently, it is unknown whether inflammatory conditions present in an inflamed dental pulp tissue could alter the immunomodulatory properties of DPSCs. This study aimed to evaluate the immunomodulatory capacity in vitro of DPSCs derived from healthy and inflamed dental pulp.MethodsDPSCs from 10 healthy and inflamed dental pulps (irreversible pulpitis) were characterized according to the minimal criteria of the International Society for Cell Therapy, proliferation, differential potential, and colony-forming units. Furthermore, the immunomodulatory capacity of DPSCs was tested on the proliferation of T lymphocytes by flow cytometry and the in vitro enzyme activity of indoleamine 2, 3-dioxygenase.ResultsThere were no significant differences in the DPSC characteristics and properties such as immunophenotype, tridifferentiation, colony-forming units, and proliferation of the DPSCs derived from normal and inflamed pulp tissue. Furthermore, there were significant differences in the immunomodulatory capacity of DPSCs obtained from human healthy dental pulp and with the diagnosis of irreversible pulpitis.ConclusionsOur results showed that DPSCs isolated from inflamed dental pulp showed typical characteristics of MSCs and diminished immunosuppressive capacity in vitro in comparison with MSCs derived from healthy dental pulp. Further investigation in vivo is needed to clarify the mechanism of this diminished immunosuppressive capacity.