Stem Cell-based Tissue Engineering Research Articles

Identification and Characterizations of Annulus Fibrosus-Derived Stem Cells.

Annulus fibrosus (AF) injuries are common in degenerative disc disease (DDD) and can lead to substantial deterioration of the intervertebral disc. However, repair or regeneration of AF remains challenging. Recently, we have found that there exists a subpopulation of cells, which form colonies in vitro and could self-renew, in AF tissue. These cells express typical surface antigen molecules of mesenchymal stem cells, including CD29, CD44, and CD166. They also express common stem cell markers such as Oct-4, nucleostemin, and SSEA-4. In addition, they can be induced to differentiate into adipocytes, osteocytes, and chondrocytes. Being AF tissue-specific, such AF-derived stem cells may potentially be an ideal candidate for DDD treatments using stem cell-based cell therapies or tissue engineering approaches.

Induction of Rhesus Keratinocytes into Functional Ameloblasts by Mouse Embryonic Dental Mesenchyme.

Fast progresses in stem cell-based tooth tissue engineering have been achieved in recent years in several animal models including the mouse, rat, dog, and pig. Moreover, various postnatal mesenchymal stem cells of dental origin have been isolated and shown capable of differentiating into odontoblasts and generating dentin. Meanwhile, human keratinocyte stem/progenitor cells, gingival epithelial cells, and even iPSC-derived epithelium have been demonstrated to be able to differentiate into functional ameloblasts. Translational medicine studies in the nonhuman primate are irreplaceable steps towards clinical application of stem cell-based tissue engineering therapy. In the present study, we first examined the epithelial stem cell markers in the rhesus skin using immunostaining. Keratinocyte stem cells were then isolated from rhesus epidermis, cultured in vitro, and characterized by epithelial stem cell markers. Epithelial sheets of these cultured keratinocytes, which were recombined with E13.5 mouse dental mesenchyme that possesses odontogenic potential in the presence of exogenous FGF8, were induced to differentiate into enamel-secreting ameloblasts. Our results demonstrate that in the presence of appropriate odontogenic signals, rhesus keratinocytes can be induced to gain odontogenic competence and are capable of participating in odontogenesis, indicating that rhesus keratinocytes are an ideal epithelial cell source for further translational medicine study of tooth tissue engineering in nonhuman primates.

Regulating osteogenesis and adipogenesis in adipose-derived stem cells by controlling underlying substrate stiffness.

Cells reside in a complex microenvironment (niche) in which the biochemical and biophysical properties of the extracellular matrix profoundly affect cell behavior. Extracellular stiffness, one important bio-mechanical characteristic of the cell niche, is important in regulating cell proliferation, migration, and lineage specification. However, the mechanism by which mechanical signals guide osteogenic and adipogenic commitment of stem cells remains difficult to dissect. To explore this question, we generated a range of polydimethylsiloxane-based matrices with differing degrees of stiffness that mimicked the stiffness seen in natural tissues and examined adipose stem cell morphology, spreading, vinculin expression, and differentiation along the osteogenic and adipogenic pathways. Rigid matrices allowed broader cell spreading, faster growth rate and stronger expression of vinculin in adipose-derived stem cells. In the presence of inductive culture media, stiffness-dependent osteogenesis and adipogenesis of the adipose stem cells indicated that there was a combinatorial effect of biophysical and biochemical cues; no such lineage specification was observed in normal media. Osteogenic differentiation behavior showed a correlation with matrix rigidity, as well as with elevated expression of RhoA, ROCK-1/-2, and related proteins in the Wnt/β-catenin pathway. The result provides a comprehensive understanding of how stem cells respond to the surrounding microenvironment and points to the fact that matrix stiffness is a critical element in biomaterial design and this will be an important advance in stem cell-based tissue engineering.

Hyaluronan size alters chondrogenesis of adipose-derived stem cells via the CD44/ERK/SOX-9 pathway.

HA-based biomaterials have been studied in stem cell-based articular cartilage tissue engineering. However, little is known about the optimal HA size for stem cell chondrogenesis and the mechanism of how HA size modulates stem cell chondrogenesis. Accordingly, we used HAs with various Mws (80-2000 kDa) as culture substrates and tested their chondrogenic effect on ADSCs. Our results demonstrated that HAs with a Mw of 2000 kDa showed the optimal effect for chondrogenesis of ADSCs. Moreover, we found that HA size can regulate ADSC chondrogenesis via the CD44/ERK/SOX-9 pathway. This finding provides new information regarding the biochemical control of chondrogenesis by HA substrates that may add value to the development of HA-based biomaterials for articular cartilage regeneration.

Synergistic effects on mesenchymal stem cell-based cartilage regeneration by chondrogenic preconditioning and mechanical stimulation

BackgroundMesenchymal stem cells (MSCs) hold promising translational potential in cartilage regeneration. However, the efficacy of MSC-based tissue engineering is not satisfactory in the treatment of cartilage defect because of the inevitable cellular functional changes during ex vivo cell expansion. How to maintain the chondrogenic capacity of MSCs to improve their therapeutic outcomes remains an outstanding question.MethodsBone marrow-derived MSCs were firstly primed in chondrogenic induction medium which was then replaced with normal growth medium to attain the manipulated cells (M-MSCs). Methacrylated hyaluronic acid (MeHA) was synthesized as a scaffold to encapsulate the cells. The MSC- or M-MSC-laden constructs were treated with dynamic compressive loading (DL) in a bioreactor or with free loading (FL) for 14 days. Afterwards, the constructs were implanted in nude mice or rat models of osteochondral defects to test their efficiency in cartilage regeneration or repair.ResultsData showed that the resulting M-MSCs exhibited superior chondrogenic differentiation potential and survivability compared with untreated MSCs. More importantly, we found that DL significantly promoted neocartilage formation in the MeHA hydrogel encapsulated with M-MSCs after 30 days of implantation in nude mice. Furthermore, the constructs laden with M-MSCs after DL for 14 days significantly enhanced cartilage healing in a rat model of osteochondral defect.ConclusionsFindings from this study highlight the importance of maintaining chondrogenic potential of MSCs by in-vitro chondrogenic preconditioning and a synergistic effect of mechanical stimulation in cartilage engineering, which may shed light on the stem cell-based tissue engineering for cartilage repair.

Commercializing Electrospun Scaffolds For Pluripotent Stem Cell-Based Tissue Engineering Applications

Abstract Tissue engineering, the process of combining bioactive scaffolds often with cells to produce replacements for damaged organs, represents an enormous market opportunity. This review critically evaluates the commercialization potential of electrospun scaffolds for applications in stem cell biology, including tissue engineering. First, it provides an overview of pluripotent stem cells (PSCs) and their defining properties, pluripotency and the ability to self-renew. These cells serve as an important tool for engineering tissues, including for clinical applications. Next, we review the technique of electrospinning and its promise for fabricating cell culture substrates and scaffolds for directing tissue formation from stem cells and compare these scaffolds to existing technologies, such as hydrogels. We address the associated market for electrospun scaffolds for PSCs and its potential for growth along with highlighting the importance of 3D cell culture substrates for PSCs by analyzing the net capital invested in this market and the associated growth rate. This review finishes by detailing the current state of commercializing electrospun scaffolds along with pathways for translating these scaffolds from research laboratories into successful start-up companies and the associated challenges with this process.

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.

Drug depot-anchoring hydrogel: A self-assembling scaffold for localized drug release and enhanced stem cell differentiation

Localized and long-term delivery of growth factors has been a long-standing challenge for stem cell-based tissue engineering. In the current study, a polymeric drug depot-anchoring hydrogel scaffold was developed for the sustained release of macromolecules to enhance the differentiation of stem cells. Self-assembling peptide (RADA16)-modified drug depots (RDDs) were prepared and anchored to a RADA16 hydrogel. The anchoring effect of RADA16 modification on the RDDs was tested both in vitro and in vivo. It was shown that the in vitro leakage of RDDs from the RADA16 hydrogel was significantly less than that of the unmodified drug depots (DDs). In addition, the in vivo retention of injected hydrogel-incorporated RDDs was significantly longer than that of hydrogel-incorporated unmodified DDs. A model drug, vascular endothelial growth factor (VEGF), was encapsulated in RDDs (V-RDDs) as drug depot that was then anchored to the hydrogel. The release of VEGF could be sustained for 4weeks. Endothelial progenitor cells (EPCs) were cultured on the V-RDDs-anchoring scaffold and enhanced cell proliferation and differentiation were observed, compared with a VEGF-loaded scaffold. Furthermore, this scaffold laden with EPCs promoted neovascularization in an animal model of hind limb ischemia. These results demonstrate that self-assembling hydrogel-anchored drug-loaded RDDs are promising for localized and sustained drug release, and can effectively enhance the proliferation and differentiation of resident stem cells, thus lead to successful tissue regeneration.

Tenogenic Differentiation of Human Embryonic Stem Cells.

Tendon healing is complex to manage because of the limited regeneration capacity of tendon tissue; stem cell-based tissue engineering approaches may provide alternative healing strategies. We sought to determine whether human embryonic stem cells (hESC) could be induced to differentiate into tendon-like cells by the addition of exogenous bone morphogenetic protein (BMP)12 (growth differentiation factor[GDF]7) and BMP13 (GDF6). hESC (SHEF-1) were maintained with or without BMP12/13 supplementation, or supplemented with BMP12/13 and the Smad signaling cascade blocking agent, dorsomorphin. Primary rat tenocytes were included as a positive control in immunocytochemistry analysis. A tenocyte-like elongated morphology was observed in hESC after 40-days continuous supplementation with BMP12/13 and ascorbic acid (AA). These cells displayed a tenomodulin expression pattern and morphology consistent with that of the primary tenocyte control. Analysis of tendon-linked gene transcription in BMP12/13 supplemented hESC demonstrated consistent expression of COL1A2, COL3A1, DCN, TNC, THBS4, and TNMD levels. Conversely, when hESCs were cultured in the presence of BMP12/13 and dorsomorphin COL3A1, DCN, and TNC gene expression and tendon matrix formation were inhibited. Taken together, we have demonstrated that hESCs are responsive to tenogenic induction via BMP12/13 in the presence of AA. The directed in vitro generation of tenocytes from pluripotent stem cells may facilitate the development of novel repair approaches for this difficult to heal tissue.

The decellularized extracellular matrix in regenerative medicine.

The decellularized extracellular matrix in regenerative medicine.

Identification and characterization of adult mouse meniscus stem/progenitor cells

ABSTRACTMeniscal damage is a common problem that accelerates the onset of knee osteoarthritis. Stem cell-based tissue engineering treatment approaches have shown promise in preserving meniscal tissue and restoring meniscal function. The purpose of our study was to identify meniscus-derived stem/progenitor cells (MSPCs) from mouse, a model system that allows for in vivo analysis of the mechanisms underlying meniscal injury and healing. MSPCs were isolated from murine menisci grown in explant culture and characterized for stem cell properties. Flow cytometry was used to detect the presence of surface antigens related to stem cells, and qRT-PCR was used to examine the gene expression profile of MSPCs. Major proteins associated with MSPCs were localized in the adult mouse knee using immunohistochemistry. Our data show that MSPCs have universal stem cell-like properties including clonogenicity and multi-potentiality. MSPCs expressed the mesenchymal stem cell markers CD44, Sca-1, CD90, and CD73 and when cultured had elevated levels of biglycan and collagen type I, important extracellular matrix components of adult meniscus. MSPC also expressed significant levels of Lox and Igf-1, genes associated with the embryonic meniscus. Localization studies showed staining for these same proteins in the superficial and outer zones of the adult mouse meniscus, regions thought to harbor endogenous repair cells. MSPCs represent a novel resident stem cell population in the murine meniscus. Analysis of MSPCs in mice will allow for a greater understanding of the cell biology of the meniscus, essential information for enhancing therapeutic strategies for treating knee joint injury and disease.

MicroRNA-214 Suppresses Osteogenic Differentiation of Human Periodontal Ligament Stem Cells by Targeting ATF4.

Periodontitis is the main cause of adult tooth loss. Stem cell-based tissue engineering has become a promising therapy for periodontitis treatment. To date, human periodontal ligament stem cells (hPDLSCs) have been shown to be a favorable source for tissue engineering, but modulatory mechanisms of hPDLSCs remain unclear. Approximately 60% of mammalian genes are the targets of over 2000 miRNAs in multiple human cell types, and miRNAs are able to influence various biological processes in the human body, including bone formation. In this study, we found that after osteogenic induction, miR-214 was significantly decreased in hPDLSCs; therefore, we examined the effects of miR-214 on osteogenic differentiation. Computational miRNA target prediction analyses and luciferase reporter assays revealed that activating transcription factor 4 (ATF4) is a direct target of miR-214. We prepared cells overexpressing miR-214 and found that miR-214 negatively regulates osteogenic differentiation of hPDLSCs. For the target of miR-214, ATF4 protein expression level was decreased after induction. In conclusion, we found that miR-214-ATF4 axis is a novel pathway for regulating hPDLSC osteogenic differentiation.

Adipose-Derived Stem Cells Cocultured with Chondrocytes Promote the Proliferation of Chondrocytes.

Articular cartilage injury and defect caused by trauma and chronic osteoarthritis vascularity are very common, while the repair of injured cartilage remains a great challenge due to its limited healing capacity. Stem cell-based tissue engineering provides a promising treatment option for injured articular cartilage because of the cells potential for multiple differentiations. However, its application has been largely limited by stem cell type, number, source, proliferation, and differentiation. We hypothesized that (1) adipose-derived stem cells are ideal seed cells for articular cartilage repair because of their accessibility and abundance and (2) the microenvironment of articular cartilage could induce adipose-derived stem cells (ADSCs) to differentiate into chondrocytes. In order to test our hypotheses, we isolated stem cells from rabbit adipose tissues and cocultured these ADSCs with rabbit articular cartilage chondrocytes. We found that when ADSCs were cocultured with chondrocytes, the proliferation of articular cartilage chondrocytes was promoted, the apoptosis of chondrocytes was inhibited, and the osteogenic and chondrogenic differentiation of ADSCs was enhanced. The study on the mechanism of this coculture system indicated that the role of this coculture system is similar to the function of TGF-β1 in the promotion of chondrocytes.

Surface-Functionalized Silk Fibroin Films as a Platform To Guide Neuron-like Differentiation of Human Mesenchymal Stem Cells.

Surface interactions at the biomaterial-cellular interface determine the proliferation and differentiation of stem cells. Manipulating such interactions through the surface chemistry of scaffolds renders control over directed stem cell differentiation into the cell lineage of interest. This approach is of central importance for stem cell-based tissue engineering and regenerative therapy applications. In the present study, silk fibroin films (SFFs) decorated with integrin-binding laminin peptide motifs (YIGSR and GYIGSR) were prepared and employed for in vitro adult stem cell-based neural tissue engineering applications. Functionalization of SFFs with short peptides showcased the peptide sequence and nature of functionalization-dependent differentiation of bone marrow-derived human mesenchymal stem cells (hMSCs). Intriguingly, covalently functionalized SFFs with GYIGSR hexapeptide (CL2-SFF) supported hMSC proliferation and maintenance in an undifferentiated pluripotent state and directed the differentiation of hMSCs into neuron-like cells in the presence of a biochemical cue, on-demand. The observed morphological changes were further corroborated by the up-regulation of neuronal-specific marker gene expression (MAP2, TUBB3, NEFL), confirmed through semiquantitative reverse-transcription polymerase chain reaction (RT-PCR) analysis. The enhanced proliferation and on-demand directed differentiation of adult stem cells (hMSCs) by the use of an economically viable short recognition peptide (GYIGSR), as opposed to the integrin recognition protein laminin, establishes the potential of SFFs for neural tissue engineering and regenerative therapy applications.

Non-viral gene activated matrices for mesenchymal stem cells based tissue engineering of bone and cartilage.

Recent regenerative medicine and tissue engineering strategies for bone and cartilage repair have led to fascinating progress of translation from basic research to clinical applications. In this context, the use of gene therapy is increasingly being considered as an important therapeutic modality and regenerative technique. Indeed, in the last 20 years, nucleic acids (plasmid DNA, interferent RNA) have emerged as credible alternative or complement to proteins, which exhibited major issues including short half-life, loss of bioactivity in pathologic environment leading to high dose requirement and therefore high production costs. The relevance of gene therapy strategies in combination with a scaffold, following a so-called "Gene-Activated Matrix (GAM)" approach, is to achieve a direct, local and sustained delivery of nucleic acids from a scaffold to ensure efficient and durable cell transfection. Among interesting cells sources, Mesenchymal Stem Cells (MSC) are promising for a rational use in gene/cell therapy with more than 1700 clinical trials approved during the last decade. The aim of the present review article is to provide a comprehensive overview of recent and ongoing work in non-viral genetic engineering of MSC combined with scaffolds. More specifically, we will show how this inductive strategy can be applied to orient stem cells fate for bone and cartilage repair.

Histological analysis of in vitro co-culture and in vivo mice co-transplantation of stem cell-derived adipocyte and osteoblast.

Many researchers have focused on the role of adipocytes in increasing efficient bone tissue engineering and osteogenic differentiation of stem cells. Previous reports have not reached a definite consensus on whether adipocytes positively influence in vitro osteogenic differentiation and in vivo bone formation. We investigated the adipocyte influence on osteogenic differentiation from adipose-derived stromal cells (ADSCs) and bone formation through histological analysis in vitro and in vivo. Using the direct co-culture system, we analyzed the influence of adipocytes to promote the differentiation fate of ADSCs. Using co-transplantation of ADSC-derived adipocytes and osteoblasts into the dorsal region of mice, the osteogenesis and bone quality were determined by histological morphology, radiography, and the measurement of the Ca2+ concentration. The adipocyte negatively affected the osteoblast differentiation of ADSCs in the in vitro system and induced osteogenesis of osteoblasts in the in vivo system through co-transplantation. Interestingly, in the co-transplanted adipocytes and osteoblasts, the bone formation areas decreased in the osteoblast only group compared with the mixed adipocytes and osteoblast group 6 weeks after transplantation. Conversely, co-transplantation and osteoblast transplantation had similar degrees of calcification as observed from radiography analysis and the measurement of the Ca2+ concentrations. Our results revealed that adipocytes inhibited osteoblast differentiation in vitro but enhanced the efficacy of osteogenesis in vivo. In addition, the adipocytes controlled the activity of osteoclasts in the newly formed bone tissue. Our approach can be used to reconstruct bone using stem cell-based tissue engineering and to enhance the understanding of the role adipocytes play.

Nano-Enabled Approaches for Stem Cell-Based Cardiac Tissue Engineering.

Cardiac diseases are the most prevalent causes of mortality in the world, putting a major economic burden on global healthcare system. Tissue engineering strategies aim at developing efficient therapeutic approaches to overcome the current challenges in prolonging patients survival upon cardiac diseases. The integration of advanced biomaterials and stem cells has offered enormous promises for regeneration of damaged myocardium. Natural or synthetic biomaterials have been extensively used to deliver cells or bioactive molecules to the site of injury in heart. Additionally, nano-enabled approaches (e.g., nanomaterials, nanofeatured surfaces) have been instrumental in developing suitable scaffolding biomaterials and regulating stem cells microenvironment to achieve functional therapeutic outcomes. This review article explores tissue engineering strategies, which have emphasized on the use of nano-enabled approaches in combination with stem cells for regeneration and repair of injured myocardium upon myocardial infarction (MI). Primarily a wide range of biomaterials, along with different types of stem cells, which have utilized in cardiac tissue engineering will be presented. Then integration of nanomaterials and surface nanotopographies with biomaterials and stem cells for myocardial regeneration will be presented. The advantages and challenges of these approaches will be reviewed and future perspective will be discussed.

How electromagnetic fields can influence adult stem cells: positive and negative impacts.

The electromagnetic field (EMF) has a great impact on our body. It has been successfully used in physiotherapy for the treatment of bone disorders and osteoarthritis, as well as for cartilage regeneration or pain reduction. Recently, EMFs have also been applied in in vitro experiments on cell/stem cell cultures. Stem cells reside in almost all tissues within the human body, where they exhibit various potential. These cells are of great importance because they control homeostasis, regeneration, and healing. Nevertheless, stem cells when become cancer stem cells, may influence the pathological condition. In this article we review the current knowledge on the effects of EMFs on human adult stem cell biology, such as proliferation, the cell cycle, or differentiation. We present the characteristics of the EMFs used in miscellaneous assays. Most research has so far been performed during osteogenic and chondrogenic differentiation of mesenchymal stem cells. It has been demonstrated that the effects of EMF stimulation depend on the intensity and frequency of the EMF and the time of exposure to it. However, other factors may affect these processes, such as growth factors, reactive oxygen species, and so forth. Exploration of this research area may enhance the development of EMF-based technologies used in medical applications and thereby improve stem cell-based therapy and tissue engineering.

Surface functionalization of nanobiomaterials for application in stem cell culture, tissue engineering, and regenerative medicine.

Stem cell-based approaches offer great application potential in tissue engineering and regenerative medicine owing to their ability of sensing the microenvironment and respond accordingly (dynamic behavior). Recently, the combination of nanobiomaterials with stem cells has paved a great way for further exploration. Nanobiomaterials with engineered surfaces could mimic the native microenvironment to which the seeded stem cells could adhere and migrate. Surface functionalized nanobiomaterial-based scaffolds could then be used to regulate or control the cellular functions to culture stem cells and regenerate damaged tissues or organs. Therefore, controlling the interactions between nanobiomaterials and stem cells is a critical factor. However, surface functionalization or modification techniques has provided an alternative approach for tailoring the nanobiomaterials surface in accordance to the physiological surrounding of a living cells; thereby, enhancing the structural and functional properties of the engineered tissues and organs. Currently, there are a variety of methods and technologies available to modify the surface of biomaterials according to the specific cell or tissue properties to be regenerated. This review highlights the trends in surface modification techniques for nanobiomaterials and the biological relevance in stem cell-based tissue engineering and regenerative medicine. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:554-567, 2016.

In Vitro Testing of Scaffolds for Mesenchymal Stem Cell-Based Meniscus Tissue Engineering-Introducing a New Biocompatibility Scoring System.

A combination of mesenchymal stem cells (MSCs) and scaffolds seems to be a promising approach for meniscus repair. To facilitate the search for an appropriate scaffold material a reliable and objective in vitro testing system is essential. This paper introduces a new scoring for this purpose and analyzes a hyaluronic acid (HA) gelatin composite scaffold and a polyurethane scaffold in combination with MSCs for tissue engineering of meniscus. The pore quality and interconnectivity of pores of a HA gelatin composite scaffold and a polyurethane scaffold were analyzed by surface photography and Berliner-Blau-BSA-solution vacuum filling. Further the two scaffold materials were vacuum-filled with human MSCs and analyzed by histology and immunohistochemistry after 21 days in chondrogenic media to determine cell distribution and cell survival as well as proteoglycan production, collagen type I and II content. The polyurethane scaffold showed better results than the hyaluronic acid gelatin composite scaffold, with signs of central necrosis in the HA gelatin composite scaffolds. The polyurethane scaffold showed good porosity, excellent pore interconnectivity, good cell distribution and cell survival, as well as an extensive content of proteoglycans and collagen type II. The polyurethane scaffold seems to be a promising biomaterial for a mesenchymal stem cell-based tissue engineering approach for meniscal repair. The new score could be applied as a new standard for in vitro scaffold testing.