Tissue engineered scaffolds in regenerative medicine.

Stem cells are self-renewing cells that can be differentiated into other cell types. Conventional in vitro models for studying stem cells differentiation are usually preformed in two-dimensional (2D) cultures. The design of three-dimensional (3D) in vitro models which ideally are supposed to mimic the in vivo stem cells microenvironment is potentially useful for inducing stem cell derived tissue formation. Biodegradable scaffolds play an important role in creating a 3D environment to induce tissue formation. The application of scaffolding materials together with stem cell technologies are believed to hold enormous potential for tissue regeneration. In this review, we provide an overview of application of tissue engineered scaffolds and stem cells for the development of stem cell-based engineered tissue replacements. In particular, we focus on bone marrow stem cells (BMSCs) and mesenchymal stem cell (MSCs) due to their extensive clinical applications.

Patho-epidemiological features of esophageal and gastric cancers in an endemic region: a 20-year retrospective study.

BACKGROUND: Gastric and esophageal cancers are among the most lethal human malignancies worldwide. Of all malignancies estimated in Iran (47,100), gastric and esophageal cancers were responsible for 7,800 and 3,500 deaths in 2008 respectively. The present study aimed to provide an image of patho-epidemiological characteristics with their trends during two past decades with emphasis on topographic, morphologic, and some demographic features. MATERIALS AND METHODS: In a hospital-based retrospective study in 2009, all pathological reports from esophageal endoscopies and gastric biopsies through a 20 years period (1989-2008) were collected and analyzed in four interval periods (five years each). Also, all eligible samples in hospital archives were enrolled for further testing. Besides, demography, topography and morphology of all samples were determined and analyzed by statistical software. RESULTS: No significant statistical difference was seen in frequency of esophageal and gastric tumors throughout the study. Esophageal cancer cases were older than gastric. Sex ratio was 2.33/1 and men had a higher rate of both esophageal and gastric tumors. Stomach cancer included 64.3% of all cases. Inferior third and end of esophagus were common locations for esophageal tumors whereas proximal stomach was common for gastric tumors. Squamous cell carcinoma and adenocarcinoma were common morphological types of tumors in esophagus and stomach respectively. CONCLUSIONS: Morphological trends showed an increase of esophageal adenocarcinoma and diffuse/intestinal ratio in stomach cancers. Trends in incidence from gastric cancer decreased based on topographic studies but we could not find a topographical trend toward cardia.

Engineering of the embryonic and adult stem cell niches.

CONTEXT: Stem cells have the potential to generate a renewable source of cells for regenerative medicine due to their ability to self-renew and differentiate to various functional cell types of the adult organism. The extracellular microenvironment plays a pivotal role in controlling stem cell fate responses. Therefore, identification of appropriate environmental stimuli that supports cellular proliferation and lineage-specific differentiation is critical for the clinical application of the stem cell therapies. EVIDENCE ACQUISITION: Traditional methods for stem cells culture offer limited manipulation and control of the extracellular microenvironment. Micro engineering approaches are emerging as powerful tools to control stem cell-microenvironment interactions and for performing high-throughput stem cell experiments. RESULTS: In this review, we provided an overview of the application of technologies such as surface micropatterning, microfluidics, and engineered biomaterials for directing stem cell behavior and determining the molecular cues that regulate cell fate decisions. CONCLUSIONS: Stem cells have enormous potential for therapeutic and pharmaceutical applications, because they can give rise to various cell types. Despite their therapeutic potential, many challenges, including the lack of control of the stem cell microenvironment remain. Thus, a greater understanding of stem cell biology that can be used to expand and differentiate embryonic and adult stem cells in a directed manner offers great potential for tissue repair and regenerative medicine.

Toxicity evaluations of superparamagnetic iron oxide nanoparticles: cell "vision" versus physicochemical properties of nanoparticles.

In the last few decades, nanoparticles (NPs) have been recognized as promising candidates for starting a new revolution in science and technology due to their unusual properties, attracting the attention of physicists, chemists, biologists, and engineers. The aim of this study is to evaluate the toxicities (at both cellular and molecular levels) of three forms of superparamagnetic iron oxide nanoparticles (SPIONs) of various surface chemistries (COOH, plain, and NH(2)) through the comparison with gene expression patterns of three cell types (i.e., human heart, brain, and kidney). For this purpose, both an MTT assay and a DNA microarray analysis were applied in three human cell lines--HCM (heart), BE-2-C (brain), and 293T (kidney)--under the exposure to SPIONs-COOH, SPIONs-NH(2), and bare SPIONs. The specific gene alteration and hierarchical clustering revealed that SPIONs-COOH altered genes associated with cell proliferative responses due to their reactive oxygen species (ROS) properties. It was also found that the cell type can have quite a significant role in the definition of suitable pathways for detoxification of NPs, which has deep implications for the safe and high yield design of NPs for biomedical applications and will require serious consideration in the future.

Regeneration of axons in injured spinal cord by activation of bone morphogenetic protein/Smad1 signaling pathway in adult neurons.

Axon growth potential is highest in young neurons but diminishes with age, thus becoming a significant obstacle to axonal regeneration after injury in maturity. The mechanism for the decline is incompletely understood, and no effective clinical treatment is available to rekindle innate growth capability. Here, we show that Smad1-dependent bone morphogenetic protein (BMP) signaling is developmentally regulated and governs axonal growth in dorsal root ganglion (DRG) neurons. Down-regulation of the pathway contributes to the age-related decline of the axon growth potential. Reactivating Smad1 selectively in adult DRG neurons results in sensory axon regeneration in a mouse model of spinal cord injury (SCI). Smad1 signaling can be effectively manipulated by an adeno-associated virus (AAV) vector encoding BMP4 delivered by a clinically applicable and minimally invasive technique, an approach devoid of unwanted abnormalities in mechanosensation or pain perception. Importantly, transected axons are able to regenerate even when the AAV treatment is delivered after SCI, thus mimicking a clinically relevant scenario. Together, our results identify a therapeutic target to promote axonal regeneration after SCI.

Gene transfer into the lung by nanoparticle dextran-spermine/plasmid DNA complexes.

A novel cationic polymer, dextran-spermine (D-SPM), has been found to mediate gene expression in a wide variety of cell lines and in vivo through systemic delivery. Here, we extended the observations by determining the optimal conditions for gene expression of D-SPM/plasmid DNA (D-SPM/pDNA) in cell lines and in the lungs of BALB/c mice via instillation delivery. In vitro studies showed that D-SPM could partially protect pDNA from degradation by nuclease and exhibited optimal gene transfer efficiency at D-SPM to pDNA weight-mixing ratio of 12. In the lungs of mice, the levels of gene expression generated by D-SPM/pDNA are highly dependent on the weight-mixing ratio of D-SPM to pDNA, amount of pDNA in the complex, and the assay time postdelivery. Readministration of the complex at day 1 following the first dosing showed no significant effect on the retention and duration of gene expression. The study also showed that there was a clear trend of increasing size of the complexes as the amount of pDNA was increased, where the sizes of the D-SPM/pDNA complexes were within the nanometer range.

Micro and nano-scale in vitro 3D culture system for cardiac stem cells.

Despite the success to prevent or limit cardiovascular diseases, the restoration of the function of a damaged heart remains a formidable challenge. Cardiac stem cells (CSCs), with the capacity to differentiate into cardiomyocytes, hold great potential as a source of cells for regenerative medicine. A major challenge facing the clinical application of differentiated CSCs, however, is theability to generate sufficient numbers of cells with the desired phenotype. We previously established cell lines of CSCs using a c-kit antibody from adult rat hearts for use in regenerative medicine. C-kit -positive cardiac cells are well recognized as CSCs and have the potential to differentiate into cardiomyocytes. Here, before implant these cells in vivo, we first developed three-dimensional culture system (3D) using micro- and nano-scaled material. Sheets of poly(glycolic acid) (PGA) were fabricated by electrospinning. Composites of collagen-PGA were prepared that contained 0, 1.5, 3 or 6 mg of electrospun PGA nanofibers. The nanofibers were added as a sheet that formed a layer within the collagen sponge. The sponges were freeze-dried and then dehydrothermally crosslinked. A scanning electron microscopy (SEM)-based analysis of the surface of the sponges demonstrated a uniform collagenous structure regardless of the amount of PGA nanofibres included. The PGA nanofibers significantly enhanced the compressive strength of the collagen sponge. More CSCs attached to the collagen sponge incorporating 6 mg of PGA nanofibers than the sponge without PGA nanofibers. The attachment and proliferation of CSCs in the 3D culture was enhanced by incubation in a bioreactor perfusion system compared with 3D static and two-dimensional (2D; i.e. tissue culture plates) culture systems. The use of micro- and nano-scale materials in the fabrication of composites together with a 3D culture system is a very promising way to promote the culture of stem cells. (c) 2009 Wiley Periodicals, Inc. J Biomed Mater Res, 2010.

Biodegradable polymer-metal complexes for gene and drug delivery.

The delivery of genes and drugs into cells has increasingly attracted attention for the generation of genetically engineered cells. Successful drug delivery will have enormous academic, clinical, and practical impacts on gene therapy, cell and molecular biology, pharmaceutical and food industries, and bio-production. The major aim of gene therapy is to deliver genetic materials into cells effectively, genetically modifying and repairing cell functions with the possibility of inducing therapeutic healing of disease. The genetic material includes DNA, RNA, antisense, decoy DNA, and ribozymes. The aim is that the appropriate transfection would allow diseased cells to return to a healthy condition. The genetic manipulation is often manifested in the mechanisms of intracellular actions of genes and proteins, and may play an important role in making clear the key genes associated with various diseases. Based on fundamental and scientific knowledge, the delivery technology of genetic material should be applicable to producing various proteins of pharmaceutical value (e.g. cytokines, growth factors, and antibodies) and also to producing seeds resistant to harmful insects and cold weather damage. This implies that the cells might be enhanced to produce valuable pharmaceutical and food products. For each approach, it is important, for successful gene expression, to select an appropriate gene to be delivered as well as to develop the gene delivery technology to enhance transfection efficiency. This review will provide an overview of the enhanced gene expression of plasmid DNA complexed with new non-viral gene delivery vehicles by biodegradable biopolymer-metal complex, introducing our recent research data to emphasize the technical feasibility of biopolymer-metal complexes in gene therapy and biotechnology.

Quantitative analysis of cell adhesion on aligned micro- and nanofibers.

In this study, we quantitatively analyzed the affinity of cell adhesion to aligned nanofibers composed of composites of poly(glycolic acid) (PGA) and collagen. Electrospun composite fibers were fabricated at various PGA/collagen weight mixing ratio (7, 18, 40, 67, and 86%) to generate fibers that ranged in diameter from 10 mum to 500 nm. Scanning electron microscopy (SEM) observation revealed that the PGA/collagen fibers were long and uniformly aligned, irrespective of the PGA/collagen weight mixing ratio. In addition, it was observed that a significantly higher number of NIH3T3 fibroblasts adhered to nanofibers with smaller diameters in comparison to fibers with larger diameters. The highest affinity of cell adhesion was observed in the PGA/collagen fibers with diameter of 500 nm and PGA/collagen weight mixing ratio of 40%. Furthermore, the adherent cells were more elongated on fibers with smaller diameters. Thus, based on the results here, PGA/collagen composite fibers are suitable for tissue culture studies and provide an attractive material for tissue engineering applications.

DNA nanoparticles encapsulated in 3D tissue-engineered scaffolds enhance osteogenic differentiation of mesenchymal stem cells.

In this study, we enhanced the expression of a plasmid DNA in mesenchymal stem cells (MSC) by the combination of three-dimensional (3D) tissue-engineered scaffold and nonviral gene carrier. To function as an enhanced delivery of plasmid DNA, acetic anhydride was reacted with polyethylenimine (PEI) to acetylate 80% of the primary and 20% of the secondary amines (PEI-Ac(80)). This acetylated PEI has been demonstrated to show enhanced gene-delivery efficiency over unmodified PEI. Collagen sponges reinforced by incorporating of poly(glycolic acid) (PGA) fibers were used as the scaffold material. DNA nanoparticles formed through simple mixing of plasmid DNA encoding bone morphogenetic protein-2 (BMP-2) and PEI-Ac(80) solutions were encapsulated within these scaffolds. MSC were seeded into each scaffold and cultured for several weeks. Within these scaffolds, the level of BMP-2 expression by transfected MSC was significantly enhanced compared to MSC transfected by DNA nanoparticles in solution (in 2D tissue culture plates). Homogeneous bone formation was histologically observed throughout the sponges seeded with transfected MSC by using DNA nanoparticles after subcutaneous implantation into the back of rats. The level of alkaline phosphatase activity and osteocalcin content at the implanted sites of sponges seeded with transfected MSC by using DNA nanoparticles were significantly higher when compared with those seeded with other agents.

Bone regeneration on a collagen sponge self-assembled peptide-amphiphile nanofiber hybrid scaffold.

The objective of this study was to create a novel approach to promote bone induction through sustained release of growth factor from a 3-dimensional (3D) hybrid scaffold. Peptide-amphiphile (PA) was synthesized by standard solid-phase chemistry that ends with the alkylation of the NH2 terminus of the peptide. Collagen sponge was reinforced by incorporation of poly(glycolic acid) (PGA) fiber. A 3D network of nanofibers was formed by mixing basic fibroblast growth factor (bFGF) suspensions with dilute aqueous solutions of PA. A hybrid scaffold was fabricated by combination of self-assembled PA nanofibers and collagen sponge reinforced with incorporation of PGA fibers. The in vitro release profile of bFGF from hybrid scaffold was investigated, and ectopic bone formation induced by the released bFGF was assessed after subcutaneous implantation of hybrid scaffold into the backs of rats. Homogeneous bone formation was histologically observed throughout the hybrid scaffolds, in marked contrast to collagen sponge-incorporated bFGF. The level of alkaline phosphatase activity and osteocalcin content at the implanted sites of hybrid scaffolds were significantly high compared with collagen sponge incorporated with bFGF. The combination of bFGF incorporated in a collagen sponge self-assembled PA nanofiber hybrid scaffold is a promising procedure to improve bone regeneration.

Trichostatin A induces myocardial differentiation of monkey ES cells.

Human embryonic stem (ES) cell lines are one of the possible sources of cardiac myocytes to be transplanted in patients with end-staged heart failure. However, prior to the application of human of ES cells for heart failure therapy, it is critical to validate their clinical use in large animals such as primates. Cynomolgus monkey ES cells have similar properties to human ES cells and can be used for primate studies. We demonstrate that 24-h stimulation by a histone deacetylase inhibitor, trichostatin A (TSA) facilitated myocardial differentiation of monkey ES cells with embryonic bodies that were seeded on gelatin-coated dishes. TSA-induced acetylating of histone-3/4 and expression of p300, one of the intrinsic histone acetyltransferases. Thus, such induction as well as inhibition of histone deacetylase may be involved in TSA-induced differentiation of cynomolgus monkey ES cells into cardiomyocytes.

Bone regeneration through controlled release of bone morphogenetic protein-2 from 3-D tissue engineered nano-scaffold.

The objective of the present study was to enhance ectopic bone formation through the controlled release of bone morphogenetic protein-2 (BMP-2) from an injectable three dimensional (3-D) tissue engineered nano-scaffold. We demonstrate that a 3-D scaffold can be formed by mixing of peptide-amphiphile (PA) aqueous solution with BMP-2 suspension. A 3-D network of nanofibers was formed by mixing BMP-2 suspensions with dilute aqueous solutions of PA. Scanning electron microscopy (SEM) observation revealed the formation of fibrous assemblies with an extremely high aspect ratio and high surface areas. In vivo release profile of BMP-2 from 3-D network of nanofibers was investigated. In addition, ectopic bone formation induced by the released BMP-2 was assessed in a rat model using histological and biochemical examinations. It was demonstrated that the injection of an aqueous solution of PA together with BMP-2 into the back subcutis of rats, resulted in the formation of a transparent 3-D hydrogel at the injected site and induced significant homogeneous ectopic bone formation around the injected site, in marked contrast to BMP-2 injection alone or PA injection alone. The combination of BMP-2-induced bone formation is a promising procedure to improve tissue regeneration.

Cognitive impairment and frontal-subcortical geriatric syndrome are associated with metabolic syndrome in a stroke-free population.

BACKGROUND: Metabolic syndrome (Met.S) consists of a conglomeration of obesity, hypertension, glucose intolerance, and dislipidemia. Frontal-subcortical geriatric syndrome (FSCS) is caused by ischemic disruption of the frontal-subcortical network. It is unknown if Met.S is associated with FSCS. METHODS: We evaluated 422 community-dwelling elderly (> or =60) in Brazil. FSCS was defined as the presence of at least one frontal release sign (grasping, palmomental, snout, or glabellar) plus coexistence of > or =3 the following criteria: (1) cognitive impairment, (2) late-onset depression, (3) neuromotor dysfunction, and (4) urgency incontinence. All values were adjusted to age and gender. RESULTS: Met.S was present in 39.3% of all subjects. Cases without any of the FSCS components represented 37.2% ('successful neuroaging' group). People with 1-3 of the FSCS components ('borderline pathological neuroaging' group) were majority (52.6%), whereas those with 4-5 of these components (FSCS group) were minority (10.2%). Met.S was significantly associated with FSCS (OR=5.9; CI: 1.5-23.4) and cognitive impairment (OR=2.2; CI: 1.1-4.6) among stroke-free subjects. Number of Met.S components explained 30.7% of the variance on the number of FSCS criteria (P<0.001). If Met.S were theoretically removed from this population, prevalence of FSCS would decline by 31.6% and that of cognitive impairment by 21.4%. CONCLUSIONS: Met.S was significantly associated with a 5.9 and 2.2 times higher chance of FSCS and cognitive impairment, respectively. Met.S might be a major determinant of 'successful' or 'pathological' neuroaging in western societies.

Bone morphogenetic protein-4 enhances cardiomyocyte differentiation of cynomolgus monkey ESCs in knockout serum replacement medium.

Despite extensive research in the differentiation of rodent ESCs into cardiomyocytes, there have been few studies of this process in primates. In this study, we examined the role of bone morphogenic protein-4 (BMP-4) to induce cardiomyocyte differentiation of cynomolgus monkey ESCs. To study the role of BMP-4, EBs were formed and cultured in Knockout Serum Replacement (KSR) medium containing BMP-4 for 8 days and subsequently seeded in gelatin-coated dishes for 20 days. It was found that ESCs differentiated into cardiomyocytes upon stimulation with BMP-4 in KSR medium, which resulted in a large fraction of beating EBs ( approximately 16%) and the upregulation of cardiac-specific proteins in a dose and time-dependent manner. In contrast, the addition of BMP-4 in FBS-containing medium resulted in a lower fraction of beating EBs ( approximately 6%). BMP-4 acted principally between mesendodermal and mesoderm progenitors and subsequently enhanced their expression. Ultrastructural observation revealed that beating EBs contained mature cardiomyocytes with sarcomeric structures. In addition, immunostaining, reverse transcription-polymerase chain reaction, and Western blotting for cardiac markers confirmed the increased differentiation of cardiomyocytes in these cultures. Moreover, electrophysiological studies demonstrated that the differentiated cardiomyocytes were electrically activated. These findings may be useful in developing effective culture conditions to differentiate cynomolgus monkey ESCs into cardiomyocytes for studying developmental biology and for regenerative medicine.

Enhanced angiogenesis through controlled release of basic fibroblast growth factor from peptide amphiphile for tissue regeneration.

In the present study, we hypothesized that a novel approach to promote vascularization would be to create injectable three-dimensional (3-D) scaffolds with encapsulated growth factor that enhance the sustained release of growth factor and induce the angiogenesis. We demonstrate that a 3-D scaffold can be formed by mixing of peptide-amphiphile (PA) aqueous solution with basic fibroblast growth factor (bFGF) suspension. PA was synthesized by standard solid phase chemistry that ends with the alkylation of the NH(2) terminus of the peptide. A 3-D network of nanofibers was formed by mixing bFGF suspensions with dilute aqueous solutions of PA. Scanning electron microscopy (SEM) observation revealed the formation of fibrous assemblies with an extremely high aspect ratio and high surface areas. In vitro and in vivo release profile of bFGF from 3-D network of nanofibers was investigated while angiogenesis induced by the released bFGF was assessed. When aqueous solution of PA was subcutaneously injected together with bFGF suspension into the back of mice, a transparent 3-D hydrogel was formed at the injected site and induced significant angiogenesis around the injected site, in marked contrast to bFGF injection alone or PA injection alone. The combination of bFGF-induced angiogenesis is a promising procedure to improve tissue regeneration.

Ectopic bone formation in collagen sponge self-assembled peptide-amphiphile nanofibers hybrid scaffold in a perfusion culture bioreactor.

The objective of this study was to enhance ectopic bone formation in a three-dimensional (3-D) hybrid scaffold in combination with bioreactor perfusion culture system. The hybrid scaffold consists of two biomaterials, a hydrogel formed through self-assembly of peptide-amphiphile (PA) with cell suspensions in media, and a collagen sponge reinforced with poly(glycolic acid) (PGA) fiber incorporation. PA was synthesized by standard solid-phase chemistry that ends with the alkylation of the NH2 terminus of the peptide. A 3-D network of nanofibers was formed by mixing cell suspensions in media with dilute aqueous solution of PA. Scanning electron microscopy (SEM) observation revealed the formation of fibrous assemblies with an extremely high aspect ratio and high surface areas. Osteogenic differentiation of mesenchymal stem cells (MSC) in the hybrid scaffold was greatly influenced by the perfusion culture method compared with static culture method. When the osteoinduction activity of hybrid scaffold was studied following the implantation into the back subcutis of rats in terms of histological and biochemical examinations, significantly homogeneous bone formation was histologically observed throughout the hybrid scaffolds when perfusion culture was used compared with static culture method. The level of alkaline phosphatase activity and osteocalcin content at the implanted sites of hybrid scaffolds were significantly high for the perfusion group compared with those in static culture method. We conclude that combination of MSC-seeded hybrid scaffold and the perfusion method was promising to enhance in vitro osteogenic differentiation of MSC and in vivo ectopic bone formation.

Osteogenic differentiation of mesenchymal stem cells in self-assembled peptide-amphiphile nanofibers.

The proliferation and differentiation of mesenchymal stem cells (MSC) was investigated in a three dimensional (3-D) network of nanofibers formed by self-assembly of peptide-amphiphile (PA) molecules. PA was synthesized by standard solid phase chemistry that ends with the alkylation of the NH(2) terminus of the peptide. The sequence of arginine-glycine-aspartic acid (RGD) was included in peptide design as well. A 3-D network of nanofibers was formed by mixing cell suspensions in media with dilute aqueous solution of PA. Scanning electron microscopy (SEM) observation revealed the formation of fibrous assemblies with an extremely high aspect ratio and high surface areas. When rat MSC were seeded into the PA nanofibers with or without RGD, larger number of cells attached was observed in the PA nanofibers including RGD. When measured to evaluate the osteogenic differentiation of MSC, the alkaline phosphatase (ALP) activity and osteocalcin content became maximum for the PA nanofibers including RGD compared with those without RGD, although both the values were significantly higher compared with those in the static tissue culture plate (2-D culture). We concluded that the attachment, proliferation, and osteogenic differentiation of MSC were influenced by PA nanofibers as the cell scaffold.