Tissue Engineering Purposes Research Articles

3D Culture Facilitates VEGF-Stimulated Endothelial Differentiation of Adipose-Derived Stem Cells.

De novo vascularization of implantable tissue and whole organ constructs has been a significant challenge in the field of tissue engineering; the use of endothelial cell populations for this task is constrained by the cell population's limited regeneration capacity and potential for loss of function. Thus, there is a need for a stem-cell population that may be induced into an endothelial cell phenotype reliably. Adipose derived stem cells (ADSCs) are multipotent cells that can be readily isolated from donor fat and may have the potential to be readily induced into endothelial cells. The ability to stimulate endothelial differentiation of these cells has been limited in standard 2D culture. We hypothesized that 3D culture would yield better differentiation. To study the influence of cell density and culture conditions on the potential of ADSCs to differentiate into an endothelial-like state, we seeded these cells types within a 3D cell-adhesive, proteolytically degradable, peptide-modified poly(ethylene-glycol) (PEG) hydrogel. ADSCs were either cultured in basal media or pro-angiogenic media supplemented with 20ng/mL of VEGF in 2D and then encapsulated at low or high densities within the PEG-based hydrogel. These encapsulated cells were maintained in either basal media or pro-angiogenic media. Cells were then isolated from the hydrogels and cultured in Matrigel to assess the potential for tubule formation. Our work shows that maintenance of ADSCs in a pro-angiogenic medium in 2D monoculture alone does not result in any CD31 expression. Furthermore, the level of CD31 expression was affected by the density of the cells encapsulated within the PEG-based hydrogel. Upon isolation of these cells, we found that these induced ADSCs were able to form tubules within Matrigel, indicative of endothelial function, while ADSCs cultured in basal medium could not. This finding points to the potential for this stem-cell population to serve as a safe and reliable source of endothelial cells for tissue engineering and regenerative medicine purposes.

Consensus QSPR modelling for the prediction of cellular response and fibrinogen adsorption to the surface of polymeric biomaterials

ABSTRACTIn the current study, we have developed predictive quantitative structure–activity relationship (QSAR) models for cellular response (foetal rate lung fibroblast proliferation) and protein adsorption (fibrinogen adsorption (FA)) on the surface of tyrosine-derived biodegradable polymers designed for tissue engineering purpose using a dataset of 66 and 40 biodegradable polymers, respectively, employing two-dimensional molecular descriptors. Best four individual models have been selected for each of the endpoints. These models are developed using partial least squares regression with a unique combination of six and four descriptors for cellular response and protein adsorption, respectively. The generated models were strictly validated using internal and external metrics to determine the predictive ability and robustness of proposed models. Subsequently, the validated individual models for each response endpoints were used for the generation of ‘intelligent’ consensus models (http://teqip.jdvu.ac.in/QSAR_Tools/DTCLab/) to improve the quality of predictions for the external data set. These models may help in prediction of virtual polymer libraries for rational design/optimization for properties relevant to biomedical applications prior to their synthesis.

Characterization of scaffolds based on chitosan and collagen with glycosaminoglycans

Scaffolds based on chitosan (CTS), collagen (Coll), and glycosaminoglycans (GAGs) cross-linked by N-(3-dimethylamino propyl)-N′-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) mixture were obtained with the use of the freeze-drying method. They were characterized by different analyses, e.g. mechanical and swelling tests, porosity, and density measurement. Moreover, the scaffolds behavior in cell culture was examined with human osteosarcoma SaOS-2 cells. The results showed that the scaffolds based on CTS, Coll, and GAGs cross-linked by EDC/NHS present physicochemical properties appropriate for biomedical purposes. They show porosity above 90% and are highly swellable. The increasing GAGs content improves the attachment and survival of cells on the obtained scaffolds. It can be assumed that scaffolds based on CTS and Coll, GAGs-enriched and cross-linked by EDC/NHS addition are biocompatible, and have properties appropriate for the tissue engineering purposes.

Bone remodeling effect of a chitosan and calcium phosphate-based composite.

Chitosan is a biocompatible polymer that has been widely studied for tissue engineering purposes. The aim of this research was to assess bone regenerative properties of an injectable chitosan and calcium phosphate-based composite and identify optimal degree of deacetylation (%DDA) of the chitosan polymer. Drill holes were generated on the left side of a mandible in Sprague-Dawley rats, and the hole was either left empty or filled with the implant. The animals were sacrificed at several time points after surgery (7–22 days) and bone was investigated using micro-CT and histology. No significant new bone formation was observed in the implants themselves at any time points. However, substantial new bone formation was observed in the rat mandible further away from the drill hole. Morphological changes indicating bone formation were found in specimens explanted on Day 7 in animals that received implant. Similar bone formation pattern was seen in control animals with an empty drill hole at later time points but not to the same extent. A second experiment was performed to examine if the %DDA of the chitosan polymer influenced the bone remodeling response. The results suggest that chitosan polymers with %DDA between 50 and 70% enhance the natural bone remodeling mechanism.

Scaffold Techniques and Designs in Tissue Engineering Functions and Purposes: A Review

In this review paper, the definition of the tissue engineering (TE) was comprehensively explored towards scaffold fabrication techniques and applications. Scaffold properties and features in TE, biological aspects, scaffold material composition, scaffold structural requirements, and old and current manufacturing technologies were reported and discussed. In almost all the reviewed reports, the TE definition denotes renewal, development, and repairs of damaged tissues caused by various factors such as disease, injury, or congenital disabilities. TE is multidisciplinary that combines biology, biochemistry, clinical medicine, and materials science whose application in cellular systems such as organ transplantation serves as a delivery vehicle for cells and drug. According to the previous literature and this review, the scaffold fabrication techniques can be classified into two main categories: conventional and modern techniques. These TE fabrication techniques are applied in the scaffold building which later on are used in tissue and organ structure. The benefits and drawbacks of each of the fabrication techniques have been described in conjunction with current areas of research devoted to deal with some of the challenges. To figure out, the highlighted aspects aimed to define the advancements and challenges that should be addressed in the scaffold design for tissue engineering. Additionally, this study provides an excellent review of original numerical approaches focused on mechanical characteristics that can be helpful in the scaffold design assessment in the analysis of scaffold parameters in tissue engineering.

Identification of Key Signaling Pathways Orchestrating Substrate Topography Directed Osteogenic Differentiation Through High-Throughput siRNA Screening

Fibrous scaffolds are used for bone tissue engineering purposes with great success across a variety of polymers with different physical and chemical properties. It is now evident that the correct degree of curvature promotes increased cytoskeletal tension on osteoprogenitors leading to osteogenic differentiation. However, the mechanotransductive pathways involved in this phenomenon are not fully understood. To achieve a reproducible and specific cellular response, an increased mechanistic understanding of the molecular mechanisms driving the fibrous scaffold mediated bone regeneration must be understood. High throughput siRNA mediated screening technology has been utilized for dissecting molecular targets that are important in certain cellular phenotypes. In this study, we used siRNA mediated gene silencing to understand the osteogenic differentiation observed on fibrous scaffolds. A high-throughput siRNA screen was conducted using a library collection of 863 genes including important human kinase and phosphatase targets on pre-osteoblast SaOS-2 cells. The cells were grown on electrospun poly(methyl methacrylate) (PMMA) scaffolds with a diameter of 0.938 ± 0.304 µm and a flat surface control. The osteogenic transcription factor RUNX2 was quantified with an in-cell western (ICW) assay for the primary screen and significant targets were selected via two sample t-test. After selecting the significant targets, a secondary screen was performed to identify osteoinductive markers that also effect cell shape on fibrous topography. Finally, we report the most physiologically relevant molecular signaling mechanisms that are involved in growth factor free, fibrous topography mediated osteoinduction. We identified GTPases, membrane channel proteins, and microtubule associated targets that promote an osteoinductive cell shape on fibrous scaffolds.

Chitosan Imparts Better Biological Properties for Poly(ε-caprolactone) Electrospun Membranes than Dexamethasone

Poly(e-caprolactone), an aliphatic polyester with biodegradability and cytocompatibility, has been used to create scaffolds for tissue engineering purposes. However, the hydrophobicity and low water absorptivity of poly(e-caprolactone) reduce cell anchorage on their membranes. Here, poly(e-caprolactone)-based scaffolds were prepared by electrospinning of poly(e-caprolactone)-chitosan blend, and poly(e-caprolactone)-dexamethasone solution. Chitosan and dexamethasone play an essential role to increase the scaffolding performance of poly(e-caprolactone)-based electrospun membranes. A poly(e-caprolactone) membrane without chitosan and dexamethasone did not provide satisfactory results to promote cell culture of adipose mesenchymal stem cells (AMSCs). Compared to the poly(e-caprolactone)-dexamethasone surface, poly(e-caprolactone)-chitosan membrane imparts better cytoskeletal reorganization, and cell spreading, increasing the strength of cell attachment. Also, poly(e-caprolactone)-chitosan composite provides strong antimicrobial activity against Pseudomonas aeruginosa (ca. 90% inhibition). Therefore, the poly(e-caprolactone)-chitosan composite is a better alternative to treat skin diseases and promote skin regeneration than conventional approaches based on dexamethasone.

Tissue-Engineered Grafts from Human Decellularized Extracellular Matrices: A Systematic Review and Future Perspectives.

Tissue engineering and regenerative medicine involve many different artificial and biologic materials, frequently integrated in composite scaffolds, which can be repopulated with various cell types. One of the most promising scaffolds is decellularized allogeneic extracellular matrix (ECM) then recellularized by autologous or stem cells, in order to develop fully personalized clinical approaches. Decellularization protocols have to efficiently remove immunogenic cellular materials, maintaining the nonimmunogenic ECM, which is endowed with specific inductive/differentiating actions due to its architecture and bioactive factors. In the present paper, we review the available literature about the development of grafts from decellularized human tissues/organs. Human tissues may be obtained not only from surgery but also from cadavers, suggesting possible development of Human Tissue BioBanks from body donation programs. Many human tissues/organs have been decellularized for tissue engineering purposes, such as cartilage, bone, skeletal muscle, tendons, adipose tissue, heart, vessels, lung, dental pulp, intestine, liver, pancreas, kidney, gonads, uterus, childbirth products, cornea, and peripheral nerves. In vitro recellularizations have been reported with various cell types and procedures (seeding, injection, and perfusion). Conversely, studies about in vivo behaviour are poorly represented. Actually, the future challenge will be the development of human grafts to be implanted fully restored in all their structural/functional aspects.

Characterization of thin gelatin hydrogel membranes with balloon properties for dynamic tissue engineering.

Cell or tissue stretching and strain are present in any in vivo environment, but is difficult to reproduce in vitro. Here, we describe a simple method for casting a thin (about 500 μm) and soft (about 0.3 kPa) hydrogel of gelatin and a method for characterizing the mechanical properties of the hydrogel simply by changing pressure with a water column. The gelatin is crosslinked with mTransglutaminase and the area of the resulting hydrogel can be increased up 13-fold by increasing the radial water pressure. This is far beyond physiological stretches observed in vivo. Actuating the hydrogel with a radial force achieves both information about stiffness, stretchability, and contractability, which are relevant properties for tissue engineering purposes. Cells could be stretched and contracted using the gelatin membrane. Gelatin is a commonly used polymer for hydrogels in tissue engineering, and the discovered reversible stretching is particularly interesting for organ modeling applications.

Evaluating Physical and Biological Characteristics of Glutaraldehyde (GA) Cross-Linked Nano-Biocomposite Bone Scaffold

In vivo stability of biomaterial-based bone scaffolds often present a significant drawback in the development of materials for tissue engineering purpose. Previously developed nanobiocomposite bone scaffold using alginate and nano cockle shell powder has shown ideal characteristics. However, it showed high degradation rate and reduced stability in an in vivo setting. In this study, we aim to observe the effect of cross-linking glutaraldehyde (GA) in three different concentrations of 0.5%, 1% and 2% during the fabrication process as a potential factor in increasing scaffold stability. Microstructure observations of scaffolds using scanning electron microscope (SEM) showed all scaffolds crossed linked with GA and control had an ideal pore size ranging from 166.8-203.5 μm. Increase in porosity compared to the control scaffolds was observed in scaffolds cross-linked with 2% GA which also presented better structural integrity as scored through semi-quantitative methods. Tested pH values during the degradation period showed that scaffolds from all groups remained within the range of 7.73-8.76. In vitro studies using osteoblast showed no significant changes in cell viability but a significant increase in ALP enzyme levels in scaffold cross-linked with 2% GA. The calcium content released from all scaffold showed significant differences within and between the groups. It can be concluded that the use of GA in the preparation stage of the scaffold did not affect the growth and proliferation of osteoblast and use of 2% GA showed improved scaffold structural integrity and porosity.

Reinforcing materials for polymeric tissue engineering scaffolds: A review.

Final purpose of tissue engineering is to regenerate or repair damaged tissues or organs. Desired repair efficacy necessitates proper physicochemical performances of scaffolds, among which adequate mechanical properties are crucial. Therefore, reinforcing tissue engineering scaffolds has been a hot research trend in recent years. In this regard, the use of some biomaterials as reinforcing materials is a longstanding area of interest. This article introduces the most popular reinforcing materials and focuses on recent advances in the use of these materials for reinforcing mechanical properties of tissue engineering scaffolds. A classification based on materials nature, for example, bioceramics, clays, carbon-based materials, and metal oxides, is provided, followed by their effects on scaffolds properties. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 107B: 1560-1575, 2019.

Novel bioactive porous starch-siloxane matrix for bone regeneration: Physicochemical, mechanical, and in vitro properties.

So far, many studies have focused on biodegradable scaffolds for tissue engineering purposes. Herein, a starch-based biodegradable scaffold was fabricated by the freeze-drying method and cross-linked using a different concentration of 3-glycidoxypropyl-trimethoxysilane (GPTMS). Field emission scanning electron microscopy (FE-SEM) micrographs indicated an interconnected porous microstructure in which the porosity decreased as a function of starch and GPTMS content. Increasing the mechanical stability and decreasing absorption capacity and biodegradation ratio affected by the higher concentration of cross-linker and the changes in structure as a function of cross-linker enhancement. Moreover, the mineralization of hybrid structures in simulated body fluid was proved by FE-SEM image and X-ray diffraction analysis. Results indicated the more GPTMS in scaffolds led to more hydroxyapatite formation. The ability of the growth and proliferation of bone marrow mesenchyme stem cells on the constructs confirmed the ability of scaffolds for bone tissue engineering applications.

Electrospinning for polymeric implants in cardiovascular applications

Abstract Electrospinning is a method for producing fibrous polymer scaffolds that can be applied in drug delivery systems as well as for polymer-based implants. Biodegradable polymers for the purpose of cardiac tissue engineering are often applied as fibrous scaffolds for morphological mimikry of natural matrices but also drugeluting approaches are very promising. Hydrolytic degradation is one of the key parameters for successful application. The focus of our investigations is on monitoring accelerated in vitro degradation of electrospun nonwoven scaffolds. In the presented study degradation of poly(Llactide) is accelerated by alkaline hydrolysis. The process is characterized by weight loss, loss of molecular mass, surface morphology and thermal behavior of nonwoven samples, showing a fast degradation of the fibrous material within two weeks.

Synthesis of an Antimicrobial Thioanthraquinone Compound to Produce Biodegradable Electrospun Mats for Tissue Engineering Purposes

In the present study for the first time in the literature, novel S-substituted bioactive anthraquinone compound were synthesized with a new, easy and less energetic reaction method (Patent Number: TR2016/19610) from 1-chloro-9,10-dihydrodiagnosisxy-anthraquinone and butyl-3-mercaptopropionate. The resultant structure present remarkable biological properties It was purified by column chromatography. All obtained structures were characterized with spectroscopic methods (NMR, MS, FT-IR, UV etc). Antimicrobial properties of bioactive compound were determined as well. The resultant thioanthraquinone compound has been synthesized for the first time in the literature and its applications as a biomaterial were discussed in the present study. Subsequently, biodegradable electrospun mats were produced via electrospinning method for their usage in treatment as a biomaterial. Structural (FTIR), morphological (FEG-SEM) biological (antimicrobial and in-vitro tests) and mechanical (tensile testing) characterizations were conducted for these nanobiomaterials. Presenting an advantage of the novel antimicrobial compound, the produced electrospun nanobiocomposites exhibited remarkable biological, mechanical properties. With a purposeful compound synthesis and a subsequent nanobiocomposite production, the obtained electrospun mats are good canditates for biomaterials for tissue engineering purposes and wound healing materials.

Calcium Orthophosphate (CaPO4) Scaffolds for Bone Tissue Engineering Applications

The chemical and structural similarities of calcium orthophosphates (abbreviated as CaPO4)to the mineral composition of natural bones and teeth have made them a good candidate for bone tissue engineering applications. Nowadays, a variety of natural or synthetic CaPO4-based biomaterials is produced and has been extensively used for dental and orthopedic applications. Despite their inherent brittleness, CaPO4 materials possess several appealing characteristics as scaffold materials. Namely, their biocompatibility and variable stoichiometry, thus surface charge density, functionality and dissolution properties, make them suitable for both drug and growth factor delivery. Therefore, CaPO4, especially hydroxyapatite (HA) and tricalcium phosphates (TCPs), have attracted a significant interest in simultaneous use as bone grafts and drug delivery vehicles. Namely, CaPO4-based three-dimensional (3D) scaffolds and/or carriers have been designed to induce bone formation and vascularization. These scaffolds are usually porous and harbor various types of drugs, biologically active molecules and/or cells. Over the past few decades, their application as bone grafts in combination with stem cells has gained much importance. This review discusses the source, manufacturing methods and advantages of using CaPO4 scaffolds for bone tissue engineering applications. Perspective future applications comprise drug delivery and tissue engineering purposes.

Biological Tissues as Active Nematic Liquid Crystals.

Live tissues can self-organize and be described as active materials composed of cells that generate active stresses through continuous injection of energy. In vitro reconstituted molecular networks, as well as single-cell cytoskeletons show that their filamentous structures can portray nematic liquid crystalline properties and can promote nonequilibrium processes induced by active processes at the microscale. The appearance of collective patterns, the formation of topological singularities, and spontaneous phase transition within the cell cytoskeleton are emergent properties that drive cellular functions. More integrated systems such as tissues have cells that can be seen as coarse-grained active nematic particles and their interaction can dictate many important tissue processes such as epithelial cell extrusion and migration as observed in vitro and in vivo. Here, a brief introduction to the concept of active nematics is provided, and the main focus is on the use of this framework in the systematic study of predominantly 2D tissue architectures and dynamics in vitro. In addition how the nematic state is important in tissue behavior, such as epithelial expansion, tissue homeostasis, and the atherosclerosis disease state, is discussed. Finally, how the nematic organization of cells can be controlled in vitro for tissue engineering purposes is briefly discussed.

Microfabrication of AngioChip, a biodegradable polymer scaffold with microfluidic vasculature.

Microengineered biomimetic systems for organ-on-a-chip or tissue engineering purposes often fail as a result of an inability to recapitulate the in vivo environment, specifically the presence of a well-defined vascular system. To address this limitation, we developed an alternative method to cultivate three-dimensional (3D) tissues by incorporating a microfabricated scaffold, termed AngioChip, with a built-in perfusable vascular network. Here, we provide a detailed protocol for fabricating the AngioChip scaffold, populating it with endothelial cells and parenchymal tissues, and applying it in organ-on-a-chip drug testing in vitro and surgical vascular anastomosis in vivo. The fabrication of the AngioChip scaffold is achieved by a 3D stamping technique, in which an intricate microchannel network can be embedded within a 3D scaffold. To develop a vascularized tissue, endothelial cells are cultured in the lumen of the AngioChip network, and parenchymal cells are encapsulated in hydrogels that are amenable to remodeling around the vascular network to form functional tissues. Together, these steps yield a functional, vascularized network in vitro over a 14-d period. Finally, we demonstrate the functionality of AngioChip-vascularized hepatic and cardiac tissues, and describe direct surgical anastomosis of the AngioChip vascular network on the hind limb of a Lewis rat model.

Evaluation of the secretion and release of vascular endothelial growth factor from two-dimensional culture and three-dimensional cell spheroids formed with stem cells and osteoprecursor cells.

Co-culture has been applied in cell therapy, including stem cells, and has been reported to give enhanced functionality. In this study, stem-cell spheroids were formed in concave micromolds at different ratios of stem cells to osteoprecursor cells, and the amount of secretion of vascular endothelial growth factor (VEGF) was evaluated. Gingiva-derived stem cells and osteoprecursor cells in the amount of 6 × 105 were seeded on a 24-well culture plate or concave micromolds. The ratios of stem cells to osteoprecursor cells included: 0:4 (group 1), 1:3 (group 2), 2:2 (group 3), 3:1 (group 4), and 4:0 (group 5). The morphology of cells in a 2-dimensional culture (groups 1-5) showed a fibroblast-like appearance. The secretion of VEGF increased with the increase in stem cells, and a statistically significant increase was noted in groups 3, 4 and 5 when compared with the media-only group (p < 0.05). Osteoprecursor cells formed spheroids in concave microwells, and no noticeable change in the morphology was noted with the increase in stem cells. Spheroids containing stem cells were positive for the stem-cell markers SSEA-4. The secretion of VEGF from cell spheroids increased with the increase in stem cells. This study showed that cell spheroids formed with stem cells and osteoprecursor cells with different ratios, using microwells, had paracrine effects on the stem cells. The secretion of VEGF increased with the increase in stem cells. This stem-cell spheroid may be applied for tissue-engineering purposes.

Rheology of magnetic alginate hydrogels

Magnetic hydrogels are becoming increasingly in demand for technical and biomedical applications, especially for tissue engineering purposes. Among them, alginate-based magnetic hydrogels emerge as one of the preferred formulations, due to the abundance, low cost, and biocompatibility of alginate polymers. However, their relatively slow gelation kinetics provokes strong particle settling, resulting in nonhomogeneous magnetic hydrogels. Here, we study magnetic hydrogels prepared by a novel two-step protocol that allows obtaining macroscopically homogeneous systems, consisting of magnetic microparticles embedded within the alginate network. We describe a comprehensive characterization (morphology, microstructure, and mechanical properties under shear stresses) of the resulting magnetic hydrogels. We pay special attention to the effects of particle volume fraction (up to 0.33) and strength of the magnetic field on the viscoelastic properties of the magnetic hydrogels. Our results indicate that magnetic hydrogels are strongly strengthened against shear stresses as magnetic particle concentration and applied field intensity increase. Finally, we report an adaptation of the two-step protocol for the injection of the magnetic hydrogels that might be adequate for implementation in vivo. Interestingly, injected magnetic hydrogels present similar morphology and mechanical properties to noninjected hydrogels. To conclude, we report magnetic alginate hydrogels with adequate homogeneity and injectability character. These characteristics, together with the broad range of their mechanical properties, make them perfect candidates for cutting-edge technology.

Synthetic Clay–based Hypoxia Mimetic Hydrogel for Pulp Regeneration: The Impact on Cell Activity and Release Kinetics Based on Dental Pulp–derived Cells In Vitro

IntroductionThixotropic synthetic clays have been successfully used for tissue engineering in regenerative medicine. The impact of these clays on the dental pulp, in particular in combination with hypoxia-based approaches using hypoxia mimetic agents (HMAs), is unknown. Our aim was to reveal the response of dental pulp–derived cells (DPCs) to a synthetic clay–based hydrogel and evaluate the release of HMAs. MethodsUsing resazurin-based toxicity assays, live-dead staining, and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide staining, the viability of human DPCs seeded onto a synthetic clay–based hydrogel of 5%–0.15% as well as onto the hydrogels loaded with the HMAs dimethyloxalylglycine (DMOG), desferrioxamine, L-mimosine, and CoCl2 was evaluated. Furthermore, supernatant of the hydrogels loaded with HMAs were generated. Vascular endothelial growth factor (VEGF) production of DPCs in response to the supernatant was measured to reveal the cellular response to the HMAs. ResultsWe found that the synthetic clay–based hydrogel did not impair the viability of DPCs. Cell monolayer and cell cluster formations were observed on the hydrogel. No significant increase of VEGF levels was observed in the supernatant when DPCs were cultured on hydrogels loaded with HMAs. Supernatant of DMOG-loaded hydrogels stimulated VEGF production in DPCs in the first hour, whereas the effect of desferrioxamine, L-mimosine, and CoCl2 did not reach a level of significance. ConclusionsThe synthetic clay–based hydrogel represents a promising biomaterial that does not induce prominent toxic effects in DPCs. It can be loaded with DMOG to induce hypoxia mimetic activity. Overall, we provided first insights into the impact of synthetic clays on DPCs for tissue engineering purposes in regenerative endodontics.