Promising Candidates For Tissue Engineering Research Articles

Immunomodulation of endothelial differentiated mesenchymal stromal cells: impact on T and NK cells.

Wharton's jelly mesenchymal stromal cells (WJ-MSCs) are promising candidates for tissue engineering, as their immunomodulatory activity allows them to escape immune recognition and to suppress several immune cell functions. To date, however, few studies have investigated the effect of differentiation of the MSCs on this immunomodulation. To address this question, we sought to determine the impact of differentiation toward endothelial cells on immunoregulation by WJ-MSCs. Following differentiation, the endothelial-like cells (ELCs) were positive for CD31, vascular endothelial cadherin and vascular endothelial growth factor receptor 2, and able to take up acetylated low-density lipoproteins. The expression of HLA-DR and CD86, which contribute to MSCs immunoprivilege, was still weak after differentiation. We then co-cultured un- and differentiated MSCs with immune cells, under conditions of both direct and indirect contact. The proliferation and phenotype of the immune cells were analyzed and the mediators secreted by both ELCs and WJ-MSCs quantified. Interleukin (IL)-6, IL-1β, prostaglandin E2 and in particular indoleamine-2,3-dioxygenase expression were upregulated in ELCs on stimulation by T and NK cells, suggesting the possible involvement of these factors in allosuppression. ELCs co-cultured with T cells were able to generate CD25(+) T cells, which were shown to be of the CD4(+)CD25(+)FoxP3(+) regulatory subset. Direct contact between NK cells and ELCs or WJ-MSCs decreased the level of NK-activating receptor natural-killer group 2, member D. Moreover, direct co-culturing with ELCs stimulates CD73 acquisition on NK cells, a mechanism which may induce adenosine secretion by the cells and lead to an immunosuppressive function. Taken together, our results show that ELCs obtained following differentiation of WJ-MSCs remain largely immunosuppressive.

Preparation and characterization of pro-angiogenic gel derived from small intestinal submucosa

Gels derived from decellularized small intestinal submucosa (SIS) have been used to repair ischemic myocardium and deliver protein drug. However, their material properties and effects on cell behavior are not well understood, in part because of the difficulty of gelling in vitro. In this study, soluble SIS matrix, which was easily handled and could effectively gel, was successfully prepared using a modified method. Fourier transform infrared spectroscopy confirmed that the SIS gel contained not only collagen but also sulfated glycosaminoglycans (sGAGs). Interestingly, the sustained release of vascular endothelial growth factor and basic fibroblast growth factor within the SIS gel was detected, and no initial burst release was observed. The SIS gel was more capable of evoking neovascularization than collagen type I gel, as determined by tube formation experiments in human umbilical vein endothelial cells, the mouse aortic ring assay, and animal experiments. The upregulated expression of kinase insert domain receptor (KDR), Notch1, and Ang2, the key genes in angiogenesis that were evaluated in HUVECs seeded on the SIS gel, confirmed that angiogenesis bioactive factors contained in the SIS gel are indeed active and effective. The SIS gel significantly promoted neovascularization compared to the collagen type I gel in vivo. Histology revealed adequate host tissue response in engraftment both types of gels. Together, these data demonstrate that the SIS gel is a promising and attractive candidate for tissue engineering, especially in promoting vessel formation. Statement of significanceThe material properties of small intestinal submucosa (SIS) gel and the effect of these properties upon cell behavior are not well understood, in part due to the difficulty of gelling in vitro. In this study, soluble SIS matrix, which was easily handled and gelled was prepared using modified method. The material properties and biocompatibility of SIS gel were explored. The sustained release of growth factors from this gel was observed along with its degradation in vitro. The results demonstrate that the SIS gel promote angiogenesis in vitro and in vivo. The SIS gel biological properties suggest that the constituent ECM molecules released from the gel remain activity. These findings suggested that the SIS gel was a promising candidate for tissue engineering, especially in promoting vessel formation.

Characterization of polycaprolactone/collagen fibrous scaffolds by electrospinning and their bioactivity

Fibrous scaffolds for tissue engineering were fabricated using collagen extracted from Nile tilapia skin and polycaprolactone (PCL) by electrospinning. The scaffolds were characterized by scanning electron microscopy (SEM), ATR-Fourier transform infrared, X-ray diffraction, and differential scanning calorimetry. Diameters of PCL/collagen fibrous scaffolds (PCFSs) decreased from 987±274 to 689±299nm with an increase in collagen content, crystallinity was low, and crystal size was small. All of the characteristic bands of PCL and collagen could be observed in PCFSs. Furthermore, PCFSs had a higher dehydration temperature (50–60°C) than native collagen (32.5°C). The ultimate tensile strength of PCFSs increased with an increase in collagen content. Circular dichroism and a degradation assay in vitro indicated that PCFSs had good stability and a low degradation rate. Cellular behavior on PCFSs was investigated by a MTT assay, SEM, and laser scanning confocal microscopy. The results indicated that the PCFSs could provide a suitable environment for the growth and viability of L929 fibroblasts, maintain good cell adhesion, and retain good biocompatibility. It implied the possibility of using PCFSs as a promising candidate for tissue engineering.

Fibrin-based interpenetrating polymer network biomaterials with tunable biodegradability

Biodegradable interpenetrating polymer networks (IPNs) were conceived. Polyvinyl alcohol (PVA) was chosen as synthetic polymer network in which serum albumin (SA) was added to allow the material forthcoming degradation by enzymes. The PVA–SA co-networks were synthesized by free radical copolymerization of polyvinyl alcohol (PVAm) and serum albumin (SAm), both previously modified with methacrylate functions. Fibrin gel which plays a key role in wound healing and hemostasis, was selected as second partner in the IPN. Fibrin/PVA–SA IPNs were synthesized rapidly through an in situ process and they are easy to handle. The homogeneous distribution of SA and fibrin in the IPN was checked by confocal laser scanning microscopy. As expected, the copolymerization of SA with PVA allows enzyme hydrolysis of both the co-networks and the corresponding IPNs with rates depending on the total protein concentration, following the Michaelis–Menten kinetic model. IPN proteolysis is therefore easily tunable. This original approach allows for the production of IPNs enzymatically degradable. Finally, these biodegradable IPNs are good supports for cell growth because their degradability enhances viability of fibroblasts cultured on their top compared to non-biodegradable material. As they mimic the physiological microenvironment of tissues, they are promising candidates for tissue engineering.

The role of reactive oxygen species in mesenchymal stem cell adipogenic and osteogenic differentiation: a review.

Mesenchymal stromal cells (MSCs) are promising candidates for tissue engineering and regenerative medicine. The multipotent stem cell component of MSC isolates is able to differentiate into derivatives of the mesodermal lineage including adipocytes, osteocytes, chondrocytes, and myocytes. Many common pathways have been described in the regulation of adipogenesis and osteogenesis. However, stimulation of osteogenesis appears to suppress adipogenesis and vice-versa. Increasing evidence implicates a tight regulation of these processes by reactive oxygen species (ROS). ROS are short-lived oxygen-containing molecules that display high chemical reactivity toward DNA, RNA, proteins, and lipids. Mitochondrial complexes I and III, and the NADPH oxidase isoform NOX4 are major sources of ROS production during MSC differentiation. ROS are thought to interact with several pathways that affect the transcription machinery required for MSC differentiation including the Wnt, Hedgehog, and FOXO signaling cascades. On the other hand, elevated levels of ROS, defined as oxidative stress, lead to arrest of the MSC cell cycle and apoptosis. Tightly regulated levels of ROS are therefore critical for MSC terminal differentiation, although the precise sources, localization, levels and the exact species of ROS implicated remain to be determined. This review provides a detailed overview of the influence of ROS on adipogenic and osteogenic differentiation in MSCs.

Tooth-derived stem cells: Update and perspectives.

Tissue engineering is an emerging field of science that focuses on creating suitable conditions for the regeneration of tissues. The basic components for tissue engineering involve an interactive triad of scaffolds, signaling molecules, and cells. In this context, stem cells (SCs) present the characteristics of self-renewal and differentiation capacity, which make them promising candidates for tissue engineering. Although they present some common markers, such as cluster of differentiation (CD)105, CD146 and STRO-1, SCs derived from various tissues have different patterns in relation to proliferation, clonogenicity, and differentiation abilities in vitro and in vivo. Tooth-derived tissues have been proposed as an accessible source to obtain SCs with limited morbidity, and various tooth-derived SCs (TDSCs) have been isolated and characterized, such as dental pulp SCs, SCs from human exfoliated deciduous teeth, periodontal ligament SCs, dental follicle progenitor cells, SCs from apical papilla, and periodontal ligament of deciduous teeth SCs. However, heterogeneity among these populations has been observed, and the best method to select the most appropriate TDSCs for regeneration approaches has not yet been established. The objective of this review is to outline the current knowledge concerning the various types of TDSCs, and discuss the perspectives for their use in regenerative approaches.

Interpenetrating polymer networks (IPN) based on gelatin/poly(ethylene glycol) dimethacrylate/clay nanocomposites: Structure–properties relationship

The effects of cross-linking sequence (simultaneous or sequential) and incorporation of exfoliated sodium-montmorillonite (Na+-MMT) nanoclay on the structure and properties of interpenetrating polymer networks (IPNs) based on gelatin/poly(ethylene glycol)dimethacrylate were studied by means of different complementary techniques. Gelatin and PEGdmA phases were cross-linked via chemical and in-situ UV curing, respectively. 2,2-dimethoxy-2-phenylacetophenone (DMPA) (1.5% w/w) was used as photo-initiator to cross-link PEGdmA. The results showed that the incorporation of small amount of Na+-MMT nanoplatelets accelerates the kinetics of chemical cross-linking of gelatin by glutaraldehyde (1.0% w/w). This led to a new hypothesis concerning the tuning structural evolution of the IPNs by the Na+-MMT content. In the case of simultaneous IPNs, in which both phases cross-linked at the same time, the accelerated cross-linking of gelatin in the presence of exfoliated sodium-montmorillonite led to increased structural homogeneity, improved mechanical and thermal properties. Incorporation of nanoclay did not show any significant effect on the structure and properties of the IPNs synthesized via sequential method in which gelatin and PEGdma phases were cross-linked separately. For the semi-IPNs, however, Na+-MMT induced macroscopic phase separation and resulted in lower mechanical properties. These results might shed light on the mechanisms underlying structure–property relationship in biohybrid IPNs based on gelatin as promising candidates for tissue engineering and drug delivery applications.

Polysaccharides derived from tragacanth as biocompatible polymers and Gels

AbstractTragacanth gum (TG) is a natural gum whose biomedical applications are limited because of the low water solubility and the possibility to form only weak water‐insoluble gels. An innovative method to produce water‐soluble tragacanth (WST) is assessed in this work. WST structural characterization indicates a high‐molecular weight polyuronic acid, which can undergo gelling by ionotropic complexation. Biological characterization shows no cytotoxicity on Hela, HepG2, and L929 cell lines. Furthermore, TG‐based and WST‐based gel beads prepared by ionic crosslinking with ferric and zinc ions are studied. Ferric WST gels exerted better cell adhesion with L929 cells than ferric alginate gels. These characteristics make WST a promising candidate for tissue engineering and drug delivery applications. © 2013 Wiley Periodicals, Inc. J. Appl. Polym. Sci., 2013

Mesenchymal stem cells and cardiac regeneration: a sophisticated approach depends on trophic effects—what's left over? Focus on “Activation of Toll-like receptor 3 amplifies mesenchymal stem cell trophic factors and enhances therapeutic potency”

application of stem cells for tissue, in particular cardiac, regeneration represents a promising field of investigation but has not achieved clinical utility ([Fig. 1][1]). From the latter point of view, Strauer and colleagues ([7][2]) in 2001 achieved an important milestone by their administration

RhoA/Rho kinase signaling regulates transforming growth factor-β1-induced chondrogenesis and actin organization of synovium-derived mesenchymal stem cells through interaction with the Smad pathway

Recent studies have suggested that synovium-derived mesenchymal stem cells (SMSCs) may be promising candidates for tissue engineering and play an important role in cartilage regeneration. However, the mechanisms of SMSC chondrogenesis remain to be identified and characterized. The aim of this study was to evaluate the activation of the RhoA/Rho kinase (ROCK) pathway, as well as the manner by which it may contribute to chondrogenesis and the actin cytoskeletal organization of rat temporomandibular SMSCs in response to transforming growth factor-β1 (TGF-β1). Primary isolated SMSCs were treated with TGF-β1, and their actin organization was examined by fluorescein isothiocyanate-phalloidin staining. The specific biochemical inhibitors, C3 transferase, Y27632 and SB431542, were employed to evaluate the function of RhoA/ROCK and Smads. The effect of C3 transferase and Y27632 on the gene expression of chondrocyte-specific markers was evaluated by quantitative real-time polymerase chain reaction. To examine the effect of Y27632 on Smad2/3 phosphorylation induced by TGF-β1, western blot analysis was also performed. The stimulation of TGF-β1 in SMSCs resulted in the activation of the RhoA/ROCK pathway and concomitantly induced cytoskeletal reorganization, which was specifically blocked by C3 transferase and Y27632. The TGF-β-induced gene expression of Sox9, type I collagen, type II collagen and aggrecan was also inhibited by both C3 transferase and Y27632, at different levels. Y27632 treatment reduced the phosphorylation of Smad2/3 in a concentration-dependent manner. These results demonstrate the RhoA/ROCK activation regulates chondrocyte-specific gene transcription and cytoskeletal organization induced by TGF-β1 by interacting with the Smad pathway. This may have significant implications for the successful utilization of SMSCs as a cell source for articular cartilage tissue engineering.

Protein adsorption and cytocompatibility of poly(L‐lactic acid) surfaces modified with biomacromolecules

AbstractSurface modification of biomaterials has been adopted over the years in order to improve their biocompatibility. In this study, heparin, hyaluronic acid and chondroitin sulfate were used to modify the surface of poly(L‐lactic acid) (PLLA) films by an entrapment method to promote its biocompatibility and to introduce natural recognition sites to the surface of PLLA films. The surface properties of PLLA films before and after modification were characterized by using water contact angle measurement, scanning electron microscope (SEM) and X‐ray photoelectron spectroscopy (XPS). Subsequently, the protein adsorption behavior of radiolabeled (125I) fibronectin (Fn) was evaluated on the modified PLLA surfaces, including adsorption kinetics, isotherm adsorption, competitive adsorption with bovine serum albumin (BSA) or serum. The cytocompatibility of the modified PLLA films was further investigated by testing osteoblasts‐like compatibility, including cell attachment, cell proliferation, alkaline phosphatase activity and cell cycle. Experimental results indicated that the hydrophilicity of the modified PLLA films was improved by the enriched biomacromolecules. In the study of protein absorption, the entrapment of the biomacromolecules on PLLA surface could decrease and reduce Fn adsorption speed and capacity, while the three biomacromolecules had almost the same adsorption capacity. Addition of BSA or serum had great effect on the adsorption of Fn on the modified PLLA surfaces. Moreover, the modifications could significantly promote osteoblasts‐like compatibility of PLLA films. The biomacromolecules modified PLLA films, combining their individual advantages such as good mechanical property and excellent cytocompatibility, are promising candidates for tissue engineering. © 2012 Wiley Periodicals, Inc. J Appl Polym Sci, 2012

3D Cell Growth and Proliferation on a RGD Functionalized Nanofibrillar Hydrogel Based on a Conformationally Restricted Residue Containing Dipeptide

Three-dimensional (3D) hydrogels incorporating a compendium of bioactive molecules can allow efficient proliferation and differentiation of cells and can thus act as successful tissue engineering scaffolds. Self-assembled peptide-based hydrogels can be worthy candidates for such applications as peptides are biocompatible, biodegradable and can be easily functionalized with desired moieties. Here, we report 3D growth and proliferation of mammalian cells (HeLa and L929) on a dipeptide hydrogel chemically functionalized with a pentapeptide containing Arg-Gly-Asp (RGD) motif. The method of functionalization is simple, direct and can be adapted to other functional moieties as well. The functionalized gel was noncytotoxic, exhibited enhanced cell growth promoting properties, and promoted 3D growth and proliferation of cells for almost 2 weeks, with simultaneous preservation of their metabolic activities. The presence of effective cell growth supporting properties in a simple and easy to functionalize dipeptide hydrogel is unique and makes it a promising candidate for tissue engineering and cell biological applications.

Stem cells derived from amniotic fluid: new potentials in regenerative medicine

Human amniotic fluid cells have been used as a diagnostic tool for the prenatal diagnosis of fetal genetic anomalies for more than 50 years. Evidence provided in the last 5 years, however, suggests that they can also harbour a therapeutic potential for human diseases, as different populations of fetal-derived stem cells have been isolated from amniotic fluid. Mesenchymal stem cells were the first to be described, which possess the higher proliferation and differentiation plasticity of adult mesenchymal stem cells and are able to differentiate towards mesodermal lineages. Amniotic fluid stem cells have more recently been isolated. They represent a novel class of pluripotent stem cells with intermediate characteristics between embryonic and adult stem cells, as they are able to differentiate into lineages representative of all three germ layers but do not form tumours when injected in vivo. These characteristics, together with the absence of ethical issues concerning their employment, suggest that stem cells present in the amniotic fluid might be promising candidates for tissue engineering and stem cell therapy of several human disorders.

Rheological characterization of the nucleus pulposus and dense collagen scaffolds intended for functional replacement

Lumbar discectomy is an effective therapy for neurological decompression in patients suffering from sciatica due to a herniated nucleus pulposus (NP). However, high numbers of patients suffering from persisting postoperative low back pain have resulted in many strategies targeting the regeneration of the NP. For successful regeneration, the stiffness of scaffolds is increasingly recognized as a potent mechanical cue for the differentiation and biosynthetic response of (stem) cells. The aim of the current study is to characterize the viscoelastic properties of the NP and to develop dense collagen scaffolds with similar properties. The scaffolds consisted of highly dense (0.5%-12%) type I collagen matrices, prepared by plastic compression. The complex modulus of the NP was 22 kPa (at 10 rad s(-1)), which should agree with a scaffold with a collagen concentration of 23%. The loss tangent, indicative of energy dissipation, is higher for the NP (0.28) than for the scaffolds (0.12) and was not dependent of the collagen density. Gamma sterilization of the scaffolds increased the shear moduli but also resulted in more brittle behavior and a reduced swelling capacity. In conclusion, by tuning the collagen density, we can approach the stiffness of the NP. Therefore, dense collagen is a promising candidate for tissue engineering of the NP that deserves further study, such as the addition of other proteins.

Mesenchymal stem cells for tissue engineering of bone: 3D-cultivation and osteogenic differentiation on mineralized collagen

Due to their plasticity and high proliferation capacity in vitro, human mesenchymal stem cells (MSC) are promising candidates for substitution of mesenchymal tissues, such as bone. According to the tissue engineering concept, combinations of cells and three dimensional scaffolds are used to replace damaged tissue. Although various attempts have been made, the optimal combination of cells and artificial scaffold has not been found so far. In this work, human MSC were isolated from bone marrow aspirates according to standard protocols and cultivated on mineralized collagen. Osteogenic differentiation was induced by medium containing dexamethasone, ascorbic acid and beta-glycerophosphate. Cell proliferation on the scaffold (WST-1 vitality assay, total protein measurement) and osteogenic differentiation (quantitative Real-Time-RT-PCR) were monitored for 24 days. Viable cells were found within the matrix throughout the cultivation period using histological and histochemical methods. Effective osteogenic differentiation could be demonstrated by the increase of expression of osteogenic marker genes (such as alkaline phosphatase) on a molecular level. Our results make the cell/matrix construct investigated in this work a promising candidate for tissue engineering of bone using mesenchymal stem cells. This has to be tested further by in vivo analysis.

Synthesis and characterization of polypyrrole-hyaluronic acid composite biomaterials for tissue engineering applications.

New tissue engineering technologies will rely on biomaterials that physically support tissue growth and stimulate specific cell functions. The goal of this study was to create a biomaterial that combines inherent biological properties which can specifically trigger desired cellular responses (e.g., angiogenesis) with electrical properties which have been shown to improve the regeneration of several tissues including bone and nerve. To this end, composites of the biologically active polysaccharide hyaluronic acid (HA) and the electrically conducting polymer polypyrrole (PP) were synthesized and characterized. Electrical conductivity of the composite biomaterial (PP/HA) was measured by a four-point probe technique, scanning electron microscopy was used to characterize surface topography, X-ray photoelectron spectroscopy and reflectance infrared spectroscopy were used to evaluate surface and bulk chemistry, and an assay with biotinylated hyaluronic acid binding protein was used to determine surface HA content. PP/HA materials were also evaluated for in vitro cell compatibility and tissue response in rats. Smooth, conductive, HA-containing PP films were produced; these films retained HA on their surfaces for several days in vitro and promoted vascularization in vivo. PP/HA composite biomaterials are promising candidates for tissue engineering and wound-healing applications that may benefit from both electrical stimulation and enhanced vascularization.