AB012.Formation of scaffold-free cell sheet with eye-related cells for ophthalmic application

The translation of current tissue engineering approaches to clinical application is somehow limited by the use of scaffolding materials.Recently a number of in vitro scaffold-free three-dimensional culture techniques have been developed.These techniques realize the assembly of tissue-like structures including but not limited to spheroids,blood vessels and cartilage.In particular,cells can now self-assemble to form planar tissue-like structures at the interface of an aqueous-two-phase system(ATPS).The unique advantage of this technique is that without a solid substrate,planar tissue-like structures can now be assembled rapidly with very simple procedures.This technique can potentially be very useful for tissue engineering in eye because of its ability to direct cells to form monolayer.In this talk,we will introduce what ATPS is and its current applications in biomedical research.We will then present an approach to assemble cell sheets in ATPS using both primary cells isolated from porcine eyes and other cell lines.The physiological relevance of these eye-related cell sheets as well as their potentials in ophthalmic research and applications will be discussed.

Use of Cryopreserved Osteogenic Matrix Cell Sheets for Bone Reconstruction

Skeletal diseases, such as nonunion and osteonecrosis, are now treatable with tissue engineering techniques. Single cell sheets called osteogenic matrix cell sheets (OMCSs) grown from cultured bone marrow-derived mesenchymal stem cells show high osteogenic potential;however, long preparation times currently limit their clinical application. Here, we report a cryopreservation OMCS transplantation method that shortens OMCS preparation time. Cryopreserved rat OMCSs were prepared using slow- and rapid-freezing methods, thawed, and subsequently injected scaffold-free into subcutaneous sites. Rapid- and slow-frozen OMCSs were also transplanted directly to the femur bone at sites of injury. Slow-freezing resulted in higher cell viability than rapid freezing, yet all two cryopreservation methods yielded OMCSs that survived and formed bone tissue. In the rapid- and slow-freezing groups, cortical gaps were repaired and bone continuity was observed within 6 weeks of OMCS transplantation. Moreover, while no significant difference was found in osteocalcin expression between the three experimental groups, the biomechanical strength of femurs treated with slow-frozen OMCSs was significantly greater than those of non-transplant at 6 weeks post-injury. Collectively, these data suggest that slow-frozen OMCSs have superior osteogenic potential and are better suited to produce a mineralized matrix and repair sites of bone injury.

Cell sheet technology for regeneration of esophageal mucosa

The progress of tissue-engineering technology has realized development of new therapies to treat various disorders by using cultured cells. Cell-and tissue-based therapies have been successfully applied to human patients, and several tissue-engineered products have been approved by the regulatory agencies and are commercially available. In the review article, we describe our experience of development and clinical application of cell sheet-based regenerative medicine.Endoscopic mucosal resection (EMR) and endoscopic submucosal dissection (ESD) have been shown to be useful for removal of gastrointestinal neoplasms with less invasiveness compared with open surgery, especially in esophageal surgery. However, postoperative inflammation and stenosis are major complications observed after intensive mucosal resection. Therefore, we have developed novel regenerative medicine to prevent such complications and promote wound healing of esophageal mucosa after EMR or ESD. Transplantable oral mucosal epithelial cell sheets were fabricated from patients' own oral mucosa. Immediately after EMR or ESD, fabricated autologous cell sheets were endoscopically transplanted to the ulcer sites. We performed a preclinical study with a canine model. In human clinical settings, cell culture and cell sheet fabrication were performed in clean rooms according to good manufacturing practice guidelines, and pharmaceutical drugs were used as supplements to culture medium in place of research regents used in animal study. We believe that cell-based regenerative medicine would be useful to improve quality of life of patients after EMR or ESD.

Bone regeneration with osteogenic matrix cell sheet and tricalcium phosphate:An experimental study in sheep

AIM To determine the effects of a cell sheet created from sheep bone marrow and tricalcium phosphate(TCP) on osteogenesis.METHODS Bone marrow cells were harvested from a sheep and cultured in a minimal essential medium(MEM) containing ascorbic acid phosphate(AscP) and dexamethasone(Dex). After 2 wk, the formed osteogenic matrix cell sheet was lifted from the culture dish using a scraper. Additionally, harvested bone marrow cells were cultured in MEM only as a negative control group, and in MEM with AscP, Dex, and β-glycerophosphate as a positive control group. For in vitro evaluation, we measured the alkaline phosphatase(ALP) activity and osteocalcin(OC) content in the media of the cultured cells from each group. For in vivo analysis, a porous TCP ceramic was used as a scaffold. We prepared an experimental group comprising TCP scaffolds wrapped with the osteogenic matrix cell sheets and a control group consisting of the TCP scaffold only. The constructs wereimplanted subcutaneously into athymic rats and the cell donor sheep, and bone formation was confirmed by histology after 4 wk.RESULTS In the in vitro part, the mean ALP activity was 0.39 ± 0.03 mg/well in the negative control group, 0.67 ± 0.04 mg/well in the sheet group, and 0.65 ± 0.07 mg/well in the positive control group. The mean OC levels were 1.46 ± 0.33 ng/well in the negative control group, 3.92 ± 0.16 ng/well in the sheet group, and 4.4 ± 0.47 ng/well in the positive control group, respectively. The ALP activity and OC levels were significantly higher in the cell sheet and positive control groups than in the negative control group(P < 0.05). There was no significant difference in ALP activity or OC levels between the cell sheet group and the positive control group(P > 0.05). TCP constructs wrapped with cell sheets prior to implantation showed bone formation, in contrast to TCP scaffolds alone, which exhibited poor bone formation when implanted, in the subcutaneous layer both in athymic rats and in the sheep. CONCLUSION This technique for preparing highly osteoinductive TCP may promote regeneration in large bone defects.

Morphology of cylindrical cell sheets with embedded contractile ring

The behavior of large deformations of cellular tissues is usually affected by the local properties of cells and their interactions,resulting in folding which acts as an important role in the embryonic development,as well as growing and spreading of a tumor,which can rapidly promote the stereo complexity of the architecture of the tissues.In the present study,a cylindrical vertex model is constructed to explore the morphology of the tubular cell sheets subject to an embedded contractile ring.It is found that an inner region of the contractile ring in equilibrium will protrude from the tube wall,and it will suddenly collapse when the contractile strength exceeds a threshold,indicating the occurrence of a bifurcation.These results on the effect of embedded contraction in the tubular shell are quite different from the planar cases,which can reveal the importance of the interaction between the geometric and material non-linearity in cylindrical geometry.The dependence of the large deformation on the bending modulus parameters and contraction strength is also analyzed for the cylindrical cell shell.

One-step fabrication of cell sheet-laden hydrogel for accelerated wound healing

Full-thickness skin wounds are have continued to be reconstructive challenges in dermal and skin appendage regeneration, and skin substitutes are promising tools for addressing these reconstructive procedures. Herein, the one-step fabrication of a cell sheet integrated with a biomimetic hydrogel as a tissue engineered skin for skin wound healing generated in one step is introduced. Briefly, cell sheets with rich extracellular matrix, high cell density, and good cell connections were integrated with biomimetic hydrogel to fabricate gel + human skin fibroblasts (HSFs) sheets and gel + human umbilical vein endothelial cells (HUVECs) sheets in one step for assembly as a cell sheet-laden hydrogel (CSH). The designed biomimetic hydrogel formed with UV crosslinking and ionic crosslinking exhibited unique properties due to the photo-generated aldehyde groups, which were suitable for integrating into the cell sheet, and ionic crosslinking reduced the adhesive force toward the substrate. These properties allowed the gel + cell sheet film to be easily released from the substrate. The cells in the harvested cell sheet maintained excellent viability, proliferation, and definite migration abilities inside the hydrogel. Moreover, the CSH was implanted into a full-thickness skin defects to construct a required dermal matrix and cell microenvironment. The wound closure rate reached 60.00 ± 6.26% on the 2nd day, accelerating mature granulation and dermis formation with skin appendages after 14 days. This project can provide distinct guidance and strategies for the complete repair and regeneration of full-thickness skin defects, and provides a material with great potential for tissue regeneration in clinical applications.

Cardiac patches made of brown adipose-derived stem cell sheets and conductive electrospun nanofibers restore infarcted heart for ischemic myocardial infarction

Cell sheet engineering has been proven to be a promising strategy for cardiac remodeling post-myocardial infarction. However, insufficient mechanical strength and low cell retention lead to limited therapeutic efficiency. The thickness and area of artificial cardiac patches also affect their therapeutic efficiency. Cardiac patches prepared by combining cell sheets with electrospun nanofibers, which can be transplanted and sutured to the surface of the infarcted heart, promise to solve this problem. Here, we fabricated a novel cardiac patch by stacking brown adipose-derived stem cells (BADSCs) sheet layer by layer, and then they were combined with multi-walled carbon nanotubes (CNTs)-containing electrospun polycaprolactone/silk fibroin nanofibers (CPSN). The results demonstrated that BADSCs tended to generate myocardium-like structures seeded on CPSN. Compared with BADSCs suspension-containing electrospun nanofibers, the transplantation of the CPSN-BADSCs sheets (CNBS) cardiac patches exhibited accelerated angiogenesis and decreased inflammation in a rat myocardial infarction model. In addition, the CNBS cardiac patches could regulate macrophage polarization and promote gap junction remodeling, thus restoring cardiac functions. Overall, the hybrid cardiac patches made of electrospun nanofibers and cell sheets provide a novel solution to cardiac remodeling after ischemic myocardial infarction.

Mechanically conditioned cell sheets cultured on thermo-responsive surfaces promote bone regeneration

Cell sheet-based scaffold-free technology holds promise for tissue engineering applications and has been extensively explored during the past decades.However,efficient harvest and handling of cell sheets remain challenging,including insufficient extracellular matrix content and poor mechanical strength.Mechanical loading has been widely used to enhance extracellular matrix production in a variety of cell types.However,currently,there are no effective ways to apply mechanical loading to cell sheets.In this study,we prepared thermo-responsive elastomer substrates by grafting poly(N-isopropyl acrylamide)(PNIPAAm)to poly(dimethylsiloxane)(PDMS)surfaces.The effect of PNIPAAm grafting yields on cell behaviours was investigated to optimize surfaces suitable for cell sheet culturing and harvesting.Subsequently,MC3T3-E1 cells were cultured on the PDMS-g-PNIPAAm substrates under mechanical stimulation by cyclically stretching the substrates.Upon maturation,the cell sheets were harvested by lowering the temperature.We found that the extracellular matrix content and thickness of cell sheet were markedly elevated upon appropriate mechanical conditioning.Reverse transcription quantitative polymerase chain reaction and Western blot analyses further confirmed that the expression of osteogenic-specific genes and major matrix components were up-regulated.After implantation into the critical-sized calvarial defects of mice,the mechanically conditioned cell sheets significantly promoted new bone formation.Findings from this study reveal that thermo-responsive elastomer,together with mechanical conditioning,can potentially be applied to prepare high-quality cell sheets for bone tissue engineering.

Recent development of temperature-responsive surfaces and their application for cell sheet engineering

Cell sheet engineering,which fabricates sheet-like tissues without biodegradable scaffolds,has been proposed as a novel approach for tissue engineering.Cells have been cultured and proliferate to confluence on a temperature-responsive cell culture surface at 37℃.By decreasing temperature to 20℃,an intact cell sheet can be harvested from the culture surface without enzymatic treatment.This new approach enables cells to keep their cell–cell junction,cell surface proteins and extracellular matrix.Therefore,recovered cell sheet can be easily not only transplanted to host tissue,but also constructed a three-dimensional(3D)tissue by layering cell sheets.Moreover,cell sheet manipulation technology and bioreactor have been combined with the cell sheet technology to fabricate a complex and functional 3D tissue in vitro.So far,cell sheet technology has been applied in regenerative medicine for several tissues,and a number of clinical studies have been performed.In this review,recent advances in the preparation of temperature-responsive cell culture surface,the fabrication of organ-like tissue and the clinical application of cell sheet engineering are summarized and discussed.

Novel pre-vascularized tissue-engineered dermis based on stem cell sheet technique used for dermis-defect healing

Insufficient donor dermis and the shortage of three-dimensional vascular networks are the main limitations in the tissue-engineered dermis(TED).To solve these problems,we initially constructed pre-vascularized bone marrow mesenchymal stem cell sheet(PBMCS)and pre-vascularized fibroblasts cell sheet(PFCS)by cell sheet technology,and then superimposed or folded them together to construct a pre-vascularized TED(PTED),aiming to mimic the real dermis structure.The constructed PTED was implanted in nude mice dorsal dermis-defect wound and the wound-healing effect was quantified at Days 1,7 and 14 via the methods of histochemistry and immunohistochemistry.The results showed that PTED could rapidly promote the wound closure,especially at Day 14,and the wound-healing rate of three-layer PTED could reach 97.2%(P<0.01),which was faster than the blank control group(89.1%),PBMCS(92.4%),PFCS(93.8%)and six-layer PTED(92.3%).In addition,the vessel density in the PTED group was higher than the other groups on the 14th day.Taken together,it is proved that the PTED,especially three-layer PTED,is more conducive to the fullthickness dermis-defect repair and the construction of the three-dimensional vascular networks,indicating its potential application in dermis-defect repair.

Cell sheet formation enhances the therapeutic effects of human umbilical cord mesenchymal stem cells on myocardial infarction as a bioactive material

Stem cell-based therapy has been used to treat ischaemic heart diseases for two decades.However,optimal cell types and transplantation methods remain unclear.This study evaluated the therapeutic effects of human umbilical cord mesenchymal stem cell(hUCMSC)sheet on myocardial infarction(MI).Methods:hUCMSCs expressing luciferase were generated by lentiviral transduction for in vivo bio-luminescent imaging tracking of cells.We applied a temperature-responsive cell culture surface-based method to form the hUCMSC sheet.Cell retention was evaluated using an in vivo bio-luminescent imaging tracking system.Unbiased transcriptional profiling of infarcted hearts and further immunohistochemical assessment of monocyte and macrophage subtypes were used to determine the mechanisms underlying the therapeutic effects of the hUCMSC sheet.Echocardiography and pathological analyses of heart sections were performed to evaluate cardiac function,angiogenesis and left ventricular remodelling.Results:When transplanted to the infarcted mouse hearts,hUCMSC sheet significantly improved the retention and survival compared with cell suspension.At the early stage of MI,hUCMSC sheet modulated inflammation by decreasing Mcp1-positive monocytes and CD68-positive macrophages and increasing Cx3cr1-positive non-classical macrophages,preserving the cardiomyocytes from acute injury.Moreover,the extracellular matrix produced by hUCMSC sheet then served as bioactive scaffold for the host cells to graft and generate new epicardial tissue,providing mechanical support and routes for revascularsation.These effects of hUCMSC sheet treatment significantly improved the cardiac function at days 7 and 28 post-MI.Conclusions:hUCMSC sheet formation dramatically improved the biological functions of hUCMSCs,mitigating adverse post-MI remodelling by modulating the inflammatory response and providing bioactive scaffold upon transplantation into the heart.Translational perspective:Due to its excellent availability as well as superior local cellular retention and survival,allogenic transplantation of hUCMSC sheets can more effectively acquire the biological functions of hUCMSCs,such as modulating inflammation and enhancing angiogenesis.Moreover,the hUCMSC sheet method allows the transfer of an intact extracellular matrix without introducing exogenous or synthetic biomaterial,further improving its clinical applicability.

Bioactive polydimethylsiloxane surface for optimal human mesenchymal stem cell sheet culture

Human mesenchymal stem cell(hMSC)sheets hold great potential in engineering three-dimensional(3D)completely biological tissues for diverse applications.Conventional cell sheet culturing methods employing thermoresponsive surfaces are cost ineffective,and rely heavily on available facilities.In this study,a cost-effective method of layer-by-layer grafting was utilized for covalently binding a homogenous collagen I layer on a commonly used polydimethylsiloxane(PDMS)substrate surface in order to improve its cell adhesion as well as the uniformity of the resulting hMSC cell sheet.Results showed that a homogenous collagen I layer was obtained via this grafting method,which improved hMSC adhesion and attachment through reliable collagen I binding sites.By utilizing this low-cost method,a uniform hMSC sheet was generated.This technology potentially allows for mass production of hMSC sheets to fulfill the demand of thick hMSC constructs for tissue engineering and biomanufacturing applications.

Regenerative medicine of cornea by cell sheet engineering using temperature-responsive culture surfaces

Recently,regenerative medicine has been focused on as next-generation definitive therapies for several diseases or injuries for which there are currently no effective treatments.These therapies have been rapidly developed through the effective fusion between different fields such as stem cell biology and biomaterials.So far,we have proposed"cell sheet engineering"through our core technology that simply applies alterations of the temperature which allows regulation of the attachment or detachment of living cells on the culture surfaces grafted with the temperature-responsive polymer"poly(N-isoproplyacrylamide)".This technology enables us to construct bioengineered sheet-like tissues,termed"cell sheets",without the need of using biodegradable scaffolds and protease treatments that are traditionally used.Therefore,our carrier-free cell sheets not only are independent of the issues observed in conventional methods,but also showed further advantages in the reconstruction of the corneal epithelium or endothelium(e.g.improvement of optical transparency and cell-cell interactions to host stroma in reconstructed tissues).Moreover,our approach does not have issues such as immune rejection or limited number of donors,due to the use of cell sheets derived from autologous limbal(in patients with unilateral disease)or oral mucosal epithelial cells(in patients with bilateral disorders).Indeed,we have successfully performed the clinical application for the reconstruction of ocular surfaces through the transplantation of our carrier-free corneal epithelial cell sheets,as evidenced by improved visual acuity as well as long-term maintenance of postoperative health conditions on ocular surfaces in all patients.We have also proposed a novel approach for the reconstruction of the corneal endothelium using corneal endothelial cell sheets by demonstrating its successful application.Thus,our cell sheet engineering technique provides a breakthrough in the field of regenerative medicine applied to the cornea.

Calculated and Experimental Research of Sheet Resistances of Laser-Doped Silicon Solar Cells

The calculated and experimental research of sheet resistances of crystalline silicon solar cells by dry laser doping is investigated. The nonlinear numerical model on laser melting of crystalline silicon and liquid-phase diffusion of phosphorus atoms by dry laser doping is analyzed by the finite difference method implemented in MATLAB. The melting period and melting depth of crystalline silicon as a function of laser energy density is achieved. The effective liquid-phase diffusion of phosphorus atoms in melting silicon by dry laser doping is confirmed by the rapid decrease of sheet resistances in experimental measurement. The plateau of sheet resistances is reached at around 15 Ω/. The calculated sheet resistances as a function of laser energy density is obtained and the calculated results are in good agreement with the corresponding experimental measurement. Due to the successful verification by comparison between experimental measurement and calculated results, the simulation results could be used to optimize the virtual laser doping parameters.

辐射合成的温敏材料上兔角膜基质细胞的培养

目的:探索以电子加速器合成具有温度敏感特性的聚异丙基丙烯酰胺凝胶(PNIPAAm)及接枝有PNIPAAm水凝胶的培养皿的方法,及兔角膜基质细胞在温敏材料PNIPAAm水凝胶上的生长条件及特性,以及利用PNIPAAm水凝胶获得的细胞片。方法:55%N-异丙基丙烯酰胺(NIPAAm)单体和0.5%的N,N’-亚甲基双丙烯酰胺的异丙醇溶液70μL,加入到35 mm培养皿上,电子加速器下辐射合成温敏材料(PNIPAAm),后续处理后,接种兔角膜基质细胞体外培养。结果:按本实验中的单体配方以及辐射合成方案,能够在培养皿表面合成PNIPAAm,角膜基质细胞能在部分接枝了PNIPAAm的培养皿上生长,并能成片脱离。结论:使用辐射合成温敏培养皿能够获得单层及多层无载体的角膜基质细胞片。

Cold stamping for AZ31B magnesium alloy sheet of cell phone house

Electric product house of magnesium alloy sheet is usually obtained by warm stamping owing to its poor plasticity and formability at room temperature.The formability of AZ31B magnesium alloy sheet can be improved by repeated unidirectional bending(RUB)process through control of(0002)basal texture.Compared with as-received sheet,the Erichsen value(IE)of the sheet underwent RUB process increases to 5.90 from 3.53 at room temperature.It is also confirmed that cell phone houses could be stamped successfully in crank press with AZ31B magnesium alloy sheets underwent RUB process.It provides an alternative to the electronics industry in the application of magnesium alloys.

New model for cardiomyocyte sheet transplantation using a virus-cell fusion technique

AIM: To facilitate close contacts between transplanted cardiomyocytes and host skeletal muscle using cell fusion mediated by hemagglutinating virus of Japan envelope(HVJ-E) and tissue maceration. METHODS: Cardiomyocytes(1.5 × 106) from fetal rats were first cultured. After proliferation, some cells were used for fusion with adult muscle fibers using HVJ-E. Other cells were used to create cardiomyocyte sheets(area: about 3.5 cm2 including 2.1 × 106 cells), which were then treated with Nile blue, separated, and transplanted between the latissimus dorsi and intercostal muscles of adult rats with four combinations of HVJ-E and/or Na OH maceration: G1: HVJ-E(+), Na OH(+), Cardiomyocytes(+); G2: HVJ-E(-), NaO H(+), Cardiomyocytes(+); G3: HVJ-E(+),Na OH(-), Cardiomyocytes(+); G4: HVJ-E(-), Na OH(-), Cardiomyocytes(-). At 1 and 2 wk after transplantation, the four groups were compared by detection of beating domains, motion images using moving target analysis software, action potentials, gene expression of MLC-2v and Mesp1 by reverse transcription-polymerase chain reaction, hematoxylin-eosin staining, and immunostaining for cardiac troponin and skeletal myosin.RESULTS: In vitro cardiomyocytes were fused with skeletal muscle fibers using HVJ-E. Cardiomyocyte sheets remained in the primary transplanted sites for 2 wk. Although beating domains were detected in G1, G2, and G3 rats, G1 rats prevailed in the number, size, motion image amplitudes, and action potential compared with G2 and G3 rats. Close contacts were only found in G1 rats. At 1 wk after transplantation, the cardiomyocyte sheets showed adhesion at various points to the myoblast layer in the latissimus dorsi muscle. At 2 wk after transplantation, close contacts were seen over a broad area. Part of the skeletal muscle sarcoplasma seemed to project into the myocardiocyte plasma and some nuclei appeared to share both sarcoplasmas.CONCLUSION: The present results show that close contacts were acquired and facilitated the beating function, thereby providing a new cellular transplantation method using HVJ-E and NaO H maceration.

心肌补片:细胞来源、完善策略及最佳制作方法分析

背景:心肌补片是一种修复损伤心肌的有效方式,但目前使用哪种细胞制作心肌补片和如何使心肌补片在体内发挥最大的治疗效果还存在争议。目的:通过综述心肌补片的细胞来源和完善心肌补片的策略来寻找出制作心肌补片的最佳方法。方法:由第一作者应用计算机以“cell sheet,cell patch,cardiomyocytes,cardia progenitor cells,fibroblasts,embryonic stem cell,mesenchymal stem cells”等为英文检索词检索PubMed和Web of Science数据库;以“心肌补片,生物3D打印,心肌”为中文检索词检索中国知网和万方数据库,经过入组筛选后,最终纳入94篇文献进入结果分析。结果与结论:①心肌补片的细胞来源主要分为3类:分别是体细胞、单能干细胞和多能干细胞。心肌补片的细胞来源丰富,但是并不是所有的细胞都适合制作心肌补片,例如,成纤维细胞和骨骼肌母细胞制成的心肌补片会有致心律失常的风险,间充质干细胞在体内的作用时间短并且存在伦理方面的问题。随着诱导式多功能干细胞的发现,为制作心肌补片提供了一个可靠的细胞来源。②心肌补片的制作方法有两种:一种是使用细胞片技术,另一种是使用生物3D打印技术。细胞片技术可完整保留细胞外基质成分,可以最大程度地模拟细胞在体内的生长循环,但是想要通过细胞片技术获得具有三维结构的心肌补片还存在困难。而生物3D打印技术可以通过计算机个性化设计获得具有三维结构的心肌补片。③完善心肌补片的策略主要包括:多种细胞共同培养后制作成心肌补片、改进生物3D打印技术中的墨水配方和支架成分、提高心肌补片的治疗效果、抑制移植后的免疫排斥反应和完善干细胞的分化培养方案。④目前还没有制作心肌补片的最佳细胞来源和制作方法,单靠某一种细胞或者某一种技术所获得的心肌补片常无法达到预期的治疗效果,因此研究者们在制作心肌补片之前需要根据预期的治疗效果来选择合适的策略制作心肌补片。

Therapeutic potential of dental pulp stem cells and their derivatives:Insights from basic research toward clinical applications

For more than 20 years,researchers have isolated and identified postnatal dental pulp stem cells(DPSCs)from different teeth,including natal teeth,exfoliated deciduous teeth,healthy teeth,and diseased teeth.Their mesenchymal stem cell(MSC)-like immunophenotypic characteristics,high proliferation rate,potential for multidirectional differentiation and biological features were demonstrated to be superior to those of bone marrow MSCs.In addition,several main application forms of DPSCs and their derivatives have been investigated,including stem cell injections,modified stem cells,stem cell sheets and stem cell spheroids.In vitro and in vivo administration of DPSCs and their derivatives exhibited beneficial effects in various disease models of different tissues and organs.Therefore,DPSCs and their derivatives are regarded as excellent candidates for stem cell-based tissue regeneration.In this review,we aim to provide an overview of the potential application of DPSCs and their derivatives in the field of regenerative medicine.We describe the similarities and differences of DPSCs isolated from donors of different ages and health conditions.The methodologies for therapeutic administration of DPSCs and their derivatives are introduced,including single injections and the transplantation of the cells with a support,as cell sheets,or as cell spheroids.We also summarize the underlying mechanisms of the regenerative potential of DPSCs.