|  |  |  | | | | | TERMSC | | | | | | | | | | | | | | | | | | | Type II collagen-hyaluronan hydrogel - a step towards a scaffold for intervertebral disc tissue engineering. Eur Cell Mater. 2010;20:134-148 Authors: Calderon L, Collin E, Velasco-Bayon D, Murphy M, O'Halloran D, Pandit A Intervertebral disc regeneration strategies based on stem cell differentiation in combination with the design of functional scaffolds is an attractive approach towards repairing/regenerating the nucleus pulposus. The specific aim of this study was to optimise a composite hydrogel composed of type II collagen and hyaluronic acid (HA) as a carrier for mesenchymal stem cells. Hydrogel stabilisation was achieved by means of 1-ethyl-3(3-dimethyl aminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS) cross-linking. Optimal hydrogel properties were determined by investigating different concentrations of EDC (8mM, 24mM and 48mM). Stable hydrogels were obtained independent of the concentration of carbodiimide used. The hydrogels cross-linked by the lowest concentration of EDC (8mM) demonstrated high swelling properties. Additionally, improved proliferation of seeded rat mesenchymal stem cells (rMSCs) and hydrogel stability levels in culture were observed with this 8mM cross-linked hydrogel. Results from this study indicate that EDC/NHS (8mM) cross-linked type II collagen/HA hydrogel was capable of supporting viability of rMSCs, and furthermore their differentiation into a chondrogenic lineage. Further investigations should be conducted to determine its potential as scaffold for nucleus pulposus regeneration/repair. PMID: 20821371 [PubMed - as supplied by publisher] | | | | | | | | | | | | | | | | | | | | | | | | | Bone Tissue Engineering for Bone Defect Therapy. Handchir Mikrochir Plast Chir. 2010 Sep 6; Authors: Boos AM, Arkudas A, Kneser U, Horch RE, Beier JP In critical size bone defects resulting from failed fracture healing or pseudarthrosis surgery is usually required. In this context, autologous bone grafts and callus distraction represent the gold standard, while sometimes even vascularised bone transfer is mandatory including microsurgical techniques. The availability of donor sites for such procedures is limited and the resulting morbidity significant. Therefore, synthetic bone grafts have been developed as an alternative. They consist of a broad range of different materials such as natural and synthetic polymers, ceramic and compound materials, aiming to mimic the three-dimensional character of autografts. In addition, they may act as a delivery vehicle for growth factors, antibiotics or cells. Their main limitation has been the lack of an intrinsic blood supply, limiting the potential for transplantation. This review provides an overview of matrices, cells and other therapeutic substances in the field of bone tissue engineering. PMID: 20821364 [PubMed - as supplied by publisher] | | | | | | | | | | | | | | | | | | | | | | | | | Reverse modeling and solid free-form fabrication of sternum implant. Australas Phys Eng Sci Med. 2010 Sep 7; Authors: Stojkovic M, Milovanovic J, Vitkovic N, Trajanovic M, Grujovic N, Milivojevic V, Milisavljevic S, Mrvic S The paper presents a case where an implant for a part of the sternum (with costal cartilages) affected by cancer was created and implanted by using the specific reverse modeling method and solid free-form fabrication. The method provides surgeons with a fast and reliable tool for tissue engineering and implantation and therefore improves the quality of life for patients. Digital images of healthy sternum samples were used to develop a reverse modeling algorithm that semi-automatically generates a necessary and sufficient simplification of the tissue geometry to be fabricated in an inexpensive and applicable manner. In this particular case, the redesign of the missing part of the sternum in CAD software took three designer-hours. At the same time, the suitable simplification of the geometry affects the fabrication of simpler and less expensive casting molds. Furthermore, the core of the developed algorithm for the reverse modeling of sternum can be applied in the reverse modeling improvement of other tile (or plate-like) bones. PMID: 20821359 [PubMed - as supplied by publisher] | | | | | | | | | | | | | | | | | | | | | | | | | A microfabricated scaffold induces the spheroid formation of human bone marrow-derived mesenchymal progenitor cells and promotes efficient adipogenic differentiation. Tissue Eng Part A. 2010 Sep 6; Authors: Miyagawa Y, Okita H, Hiroyama M, Sakamoto R, Kobayashi M, Nakajima H, Katagiri YU, Fujimoto J, Hata JI, Umezawa A, Kiyokawa N Here, we report the highly efficient in vitro differentiation of human bone marrow derived mesenchymal stem/progenitor cells (human MPCs) using a novel nanotechnology-based culture plate, Nanoculture plate(R) (NCP). The NCP contains uneven microfabrications with diameters of approximately 2-3mum arranged in a honeycomb pattern on its culture surface, which is devoid of animal-derived protein sources. When human MPCs were subjected to 3-D culture using an NCP, they rapidly formed adhesive spheroids. We showed that adipogenic differentiation in NCP-mediated 3-D cultures led to more rapid accumulation of triglycerides than that in two-dimensional (2-D) cultures. During adipogenesis in 3-D cultures, the rapid and intense induction of adipocyte-specific gene expressions, such as PPAR-gamma, C/EBP-alpha, aP2 and adiponectin was observed. Together, these results indicate that this 3-D culture system is suitable for the differentiation of human MPCs into adipogenic lineage, and could be applicable to adipose tissue engineering under xeno-free condition. PMID: 20818998 [PubMed - as supplied by publisher] | | | | | | | | | | | | | | | | | | | | | | | | | Ontogeny of the Kidney and Renal Developmental Markers in the Rhesus Monkey (Macaca Mulatta). Anat Rec (Hoboken). 2010 Sep 3; Authors: Batchelder CA, Lee CC, Martinez ML, Tarantal AF Nonhuman primates share many developmental similarities with humans, thus they provide an important preclinical model for understanding the ontogeny of biomarkers of kidney development and assessing new cell-based therapies to treat human disease. To identify morphological and developmental changes in protein and RNA expression patterns during nephrogenesis, immunohistochemistry and quantitative real-time PCR were used to assess temporal and spatial expression of WT1, Pax2, Nestin, Synaptopodin, alpha-smooth muscle actin (alpha-SMA), CD31, vascular endothelial growth factor (VEGF), and Gremlin. Pax2 was expressed in the condensed mesenchyme surrounding the ureteric bud and in the early renal vesicle. WT1 and Nestin were diffusely expressed in the metanephric mesenchyme, and expression increased as the Pax2-positive condensed mesenchyme differentiated. The inner cleft of the tail of the S-shaped body contained the podocyte progenitors (visceral epithelium) that were shown to express Pax2, Nestin, and WT1 in the early second trimester. With maturation of the kidney, Pax2 expression diminished in these structures, but was retained in cells of the parietal epithelium, and as WT1 expression was upregulated. Mature podocytes expressing WT1, Nestin, and Synaptopodin were observed from the mid-third trimester through adulthood. The developing glomerulus was positive for alpha-SMA (vascular smooth muscle) and Gremlin (mesangial cells), CD31 (glomerular endothelium), and VEGF (endothelium), and showed loss of expression of these markers as glomerular maturation was completed. These data form the basis for understanding nephrogenesis in the rhesus monkey and will be useful in translational studies that focus on embryonic stem and other progenitor cell populations for renal tissue engineering and repair. Anat Rec, 2010. (c) 2010 Wiley-Liss, Inc. PMID: 20818613 [PubMed - as supplied by publisher] | | | | | | | | | | | | | | | | | | | | | | | | | Repair of orbital wall defects using biocoral scaffolds combined with bone marrow stem cells enhanced by human bone morphogenetic protein-2 in a canine model. Int J Mol Med. 2010 Oct;26(4):517-25 Authors: Xiao C, Zhou H, Ge S, Tang T, Hou H, Luo M, Fan X Repair of orbital bone defects caused by trauma, infection or cancer is a continuous challenge in reconstructive surgery. Few studies have reported the application of tissue engineering for the repair of orbital bone defects in large animal models. Thus, we investigated the effects of tissue-engineered bone enhanced by the human bone morphogenetic protein-2 (BMP2) on the repair of orbital wall defects in a canine model. Autologous bone marrow stromal cells (BMSCs) from 16 Beagle dogs were isolated and cultured in vitro. Passage 2 cells were transfected with adenovirus containing human BMP2 (adv-BMP2) and tissue-engineered bone was constructed using BMP2-expressing BMSCs seeded on a biocoral scaffold. Circular defects (12-mm diameter) created bilaterally in the canine medial orbital wall, were treated with one of the following: adv-BMP2-transfected BMSC/coral composite (group I, n=8), BMSC/coral composite (group II, n=8), biocoral alone (group III, n=8), or were left untreated (group IV, n=8). Four samples from each group were harvested at 12 and 24 weeks after surgery, and the volume and density of newly regenerated bone were determined by micro-computed tomographic (micro-CT) measurement. The rate of new bone deposition and regeneration was measured by tetracycline/calcein labeling and histomorphometric analysis. The results showed that a canine 12-mm circular orbital defect was a critical-sized defect, and the micro-CT and histomorphometry detection results indicated that the combined delivery of BMSCs and BMP2 (group I) resulted in the highest regenerative effects on orbital bone defects, compared to the other groups without BMP2. Biocoral scaffolds combined with BMSCs enhanced by BMP2 could improve the healing of critical-sized medial orbital wall defects in canines. PMID: 20818491 [PubMed - in process] | | | | | | | | | | | | | | | | | | | | | | | | | Tumourigenesis in the infarcted rat heart is eliminated through differentiation and enrichment of the transplanted embryonic stem cells. Eur J Heart Fail. 2010 Sep 3; Authors: Lin Q, Fu Q, Zhang Y, Wang H, Liu Z, Zhou J, Duan C, Wang Y, Wu K, Wang C AIMS: The therapeutic potential of embryonic stem cells (ESCs) in ischaemic heart disease has been widely explored. However, tumourigenesis upon implantation interferes with the clinical application of ESC transplantation. This study aims to evaluate the influence of differentiation and enrichment of transplanted ESCs on tumourigenesis in infarcted rat hearts. METHODS AND RESULTS: Mouse ESCs (mESCs) were cultured using a bioreactor system to develop embryoid bodies, which were then induced with 1% ascorbic acid to differentiate into cardiomyocytes. The mESCs-derived cardiomyocytes (mESCs-CMs) were enriched by Percoll density gradient separation. The specific markers (OCT-4, Sox2, and Nanog) of undifferentiated ESCs were detected by PCR both in mESCs and in mESCs-CMs, but not in the mESC-derived Percoll-enriched cardiomyocytes (mESC-PE-CMs). Immunosuppressed rats with infarcted hearts were randomly injected with the mESCs, mESC-CMs, or mESC-PE-CMs. Eight weeks after cell transplantation, histological and immunohistochemical analysis showed that the transplantation of both mESCs and mESC-CMs caused the formation of teratomas. The incidence of teratoma was markedly lower (P < 0.05) in the mESC-CMs group than in the mESCs group. The average tumour volume was significantly lower (P < 0.05) in the mESC-CMs group than in the mESCs group. Tumour formation was absent in the mESC-PE-CMs group. CONCLUSION: Enrichment of the mESC-differentiated cardiomyocytes inhibited the development of teratoma after cell transplantation in the infarcted rat hearts. These findings offer a new strategy for eliminating teratoma formation in ESCs transplantation and could be a step forward in the development of human ESCs transplantation therapy in ischaemic heart disease. PMID: 20817694 [PubMed - as supplied by publisher] | | | | | | | | | | | | | | | | | | | | | | | | | Implantation of adult bone marrow-derived mesenchymal stem cells transfected with the neurotrophin-3 gene and pretreated with retinoic acid in completely transected spinal cord. Brain Res. 2010 Sep 2; Authors: Zhang W, Yan Q, Zeng Y, Zhang X, Yi X, Wang J, Chen S, Li Y, Bruce IC, Wu W Implantation of marrow-derived mesenchymal stem cells (MSCs) is the most promising therapeutic strategy for the treatment of spinal cord injury (SCI), especially because of their potential for clinical application, such as the avoidance of immunologic rejection, their strong secretory properties, and their plasticity for developing into neural cells. However, the recovery from SCI after MSC implantation is minimal due to their limited capacity for the reduction of cystic cavititation, for the axonal regeneration and their uncertain neural plasticity in the spinal cord. We previously pretreated MSCs with all-trans retinoic acid (RA) in vitro. Then we genetically modified them to overexpress neurotrophin-3 (NT-3) via a recombinant adenoviral vector (Adv). This combined treatment not only permitted more neuronal differentiation of MSCs, but stimulated more NT-3 secretion prior to grafting, according to our previous and present results. When these cells were implanted into the transected spinal cord of rats, the animals had some improvement (both functionally and structurally), including the recovery of hindlimb locomotor function, shown by the highest Basso, Beattie, and Bresnahan (BBB) scores, as well as dramatically reduced cavity volume, clear axonal regeneration and more neuronal survival. In contrast, simple MSC implantation is not a very effective therapy for spinal transection. However, the neuronal differentiation of MSCs after treatment with a combination of Adv-mediated NT-3 gene transfer and RA was only mildly improved in vivo. PMID: 20816761 [PubMed - as supplied by publisher] | | | | | | | | | | | | | | | | | | | | | | | | | Nonthermal irreversible electroporation for tissue decellularization. J Biomech Eng. 2010 Sep;132(9):091003 Authors: Phillips M, Maor E, Rubinsky B Tissue scaffolding is a key component for tissue engineering, and the extracellular matrix (ECM) is nature's ideal scaffold material. A conceptually different method is reported here for producing tissue scaffolds by decellularization of living tissues using nonthermal irreversible electroporation (NTIRE) pulsed electrical fields to cause nanoscale irreversible damage to the cell membrane in the targeted tissue while sparing the ECM and utilizing the body's host response for decellularization. This study demonstrates that the method preserves the native tissue ECM and produces a scaffold that is functional and facilitates recellularization. A two-dimensional transient finite element solution of the Laplace and heat conduction equations was used to ensure that the electrical parameters used would not cause any thermal damage to the tissue scaffold. By performing NTIRE in vivo on the carotid artery, it is shown that in 3 days post NTIRE the immune system decellularizes the irreversible electroporated tissue and leaves behind a functional scaffold. In 7 days, there is evidence of endothelial regrowth, indicating that the artery scaffold maintained its function throughout the procedure and normal recellularization is taking place. PMID: 20815637 [PubMed - in process] | | | | | | | | | | | | | | | | | | | | | | | | | Biocompatible, Detachable, and Free-Standing Polyelectrolyte Multilayer Films. Biomacromolecules. 2010 Sep 3; Authors: Larkin AL, Davis RM, Rajagopalan P Self-assembled polyelectrolyte multilayers have gained tremendous popularity over the past decade and have been incorporated in diverse applications. However, the fabrication of detachable and free-standing polyelectrolyte multilayers (PEMs) has proven to be difficult. We report the design of detachable, free-standing, and biocompatible PEMs comprised of hyaluronic acid (anionic PE) and chitosan (cationic PE). These PEMs can be detached from an underlying inert substrate without any postprocessing steps. Our approach enables the fabrication of detachable PEMs from a wide range of polyelectrolytes. Cross-linked PEMs exhibited greater than 95% weight retention when maintained in phosphate buffered saline at 37 degrees C over a seven day period. The PEM thickness was approximately 3 mum for dried films and increased 2-fold under hydration. A unique feature of the detachable, free-standing PEMs is their optical transparency in the 400-900 nm range under hydrated conditions. The Young's modulus of the cross-linked films ranged from 300-400 MPa, rendering these detachable free-standing multilayers ideal for biomaterial applications. BALB/c 3T3 fibroblasts adhered on the PEMs and colonized the entire surface over a six day period. The cellular responses, as well as the physical properties, demonstrate that the detachable PEM films exhibit tremendous potential for applications in biomaterials and tissue engineering. PMID: 20815399 [PubMed - as supplied by publisher] | | | | | | | | | | | | | | | | | | | | | | | | | Cryopreservation of organotypical cultures based on 3D scaffolds. Cryo Letters. 2010 Mar-Apr;31(2):157-68 Authors: Rupf T, Ebert S, Lorenz K, Salvetter J, Bader A An integral component of the manufacture of a skin substitute is the cryopreservation of the complete skin construct. Under this demand, investigations were carried out in the present work in the case of cryopreservation of human fibroblasts and keratinocytes composed to organotypical skin substitutes (OTS). Two scaffolds made up of gelatine and collagen/elastin were seeded with human fibroblasts via centrifugation method. Subsequent human keratinocytes were applied on the preceded scaffolds and cultivated under air-exposed conditions. For the investigation of the cryopreservation, OTS were frozen after 10 days cultivation via computer-controlled CryoMed included defined freezing conditions. After 24 hours storage in fluid nitrogen the OTSs were thawed and recultivated under airlift conditions. After that metabolic activity and immunfluorescent staining was analyzed in comparison with conventionally produced OTSs on basis of collagengel and/or OTSs based on scaffolds without cryopreservation. It could be assessed that cryopreservation has no negative influence on vitality and differentiation capacity of the cultivated constructs. The determination of OTS vitality after 14 days airlift culture delivered persistent higher metabolic activities of the scaffold based constructs in comparison with the corresponding controls. This could be confirmed by investigation of OTSs with and without cryopreservation. All expression patterns of differentiation marker could be detected after cryopreservation and subsequent recultivation. The results from cryopreservation of OTSs introduced here prove the possibility of temporally independent tailor-made applications by means of a complete skin substitute for example in the area pharmascreening. PMID: 20687458 [PubMed - indexed for MEDLINE] | | | | | | | | | | | | | | | | | | | | | | | | | Impaired cell surface expression of HLA-B antigens on mesenchymal stem cells and muscle cell progenitors. PLoS One. 2010;5(5):e10900 Authors: Isa A, Nehlin JO, Sabir HJ, Andersen TE, Gaster M, Kassem M, Barington T HLA class-I expression is weak in embryonic stem cells but increases rapidly during lineage progression. It is unknown whether all three classical HLA class-I antigens follow the same developmental program. In the present study, we investigated allele-specific expression of HLA-A, -B, and -C at the mRNA and protein levels on human mesenchymal stem cells from bone marrow and adipose tissue as well as striated muscle satellite cells and lymphocytes. Using multicolour flow cytometry, we found high cell surface expression of HLA-A on all stem cells and PBMC examined. Surprisingly, HLA-B was either undetectable or very weakly expressed on all stem cells protecting them from complement-dependent cytotoxicity (CDC) using relevant human anti-B and anti-Cw sera. IFNgamma stimulation for 48-72 h was required to induce full HLA-B protein expression. Quantitative real-time RT-PCR showed that IFNgamma induced a 9-42 fold increase of all six HLA-A,-B,-C gene transcripts. Interestingly, prior to stimulation, gene transcripts for all but two alleles were present in similar amounts suggesting that post-transcriptional mechanisms regulate the constitutive expression of HLA-A,-B, and -C. Locus-restricted expression of HLA-A, -B and -C challenges our current understanding of the function of these molecules as regulators of CD8(+) T-cell and NK-cell function and should lead to further inquiries into their expression on other cell types. PMID: 20531935 [PubMed - indexed for MEDLINE] | | | | | | | | | | | | | | | | | | | | | | | | | Integrin-blocking antibodies delay keratinocyte re-epithelialization in a human three-dimensional wound healing model. PLoS One. 2010;5(5):e10528 Authors: Egles C, Huet HA, Dogan F, Cho S, Dong S, Smith A, Knight EB, McLachlan KR, Garlick JA The alpha6beta4 integrin plays a significant role in tumor growth, angiogenesis and metastasis through modulation of growth factor signaling, and is a potentially important therapeutic target. However, alpha6beta4-mediated cell-matrix adhesion is critical in normal keratinocyte attachment, signaling and anchorage to the basement membrane through its interaction with laminin-5, raising potential risks for targeted therapy. Bioengineered Human Skin Equivalent (HSE), which have been shown to mimic their normal and wounded counterparts, have been used here to investigate the consequences of targeting beta4 to establish toxic effects on normal tissue homeostasis and epithelial wound repair. We tested two antibodies directed to different beta4 epitopes, one adhesion-blocking (ASC-8) and one non-adhesion blocking (ASC-3), and determined that these antibodies were appropriately localized to the basal surface of keratinocytes at the basement membrane interface where beta4 is expressed. While normal tissue architecture was not altered, ASC-8 induced a sub-basal split at the basement membrane in non-wounded tissue. In addition, wound closure was significantly inhibited by ASC-8, but not by ASC-3, as the epithelial tongue only covered 40 percent of the wound area at 120 hours post-wounding. These results demonstrate beta4 adhesion-blocking antibodies may have adverse effects on normal tissue, whereas antibodies directed to other epitopes may provide safer alternatives for therapy. Taken together, we conclude that these three-dimensional tissue models provide a biologically relevant platform to identify toxic effects induced by candidate therapeutics, which will allow generation of findings that are more predictive of in vivo responses early in the drug development process. PMID: 20502640 [PubMed - indexed for MEDLINE] | | | | | | | | | | | | | | | | | | | | | | | | | Brown adipose tissue--a new role in humans? Nat Rev Endocrinol. 2010 Jun;6(6):319-25 Authors: Lidell ME, Enerbäck S New targets for pharmacological interventions are of great importance to combat the epidemic of obesity. Brown adipose tissue could potentially represent one such target. Unlike white adipose tissue, brown adipose tissue has the ability to dissipate energy by producing heat rather than storing it as triglycerides. In small mammals, the presence of active brown adipose tissue is pivotal for the maintenance of body temperature and possibly to protect against the detrimental effects of surplus energy intake. Animal studies have shown that expansion and/or activation of brown adipose tissue counteracts diet-induced weight gain and related disorders such as type 2 diabetes mellitus. Several independent studies have now confirmed the presence of functional brown adipose tissue in adult humans, for whom this tissue is probably metabolically beneficial given its association with both low BMI and low total adipose tissue content. Over the past few years, knowledge of the transcriptional control and development of brown adipose tissue has increased substantially. Thus, several possible targets that may be useful for the expansion and/or activation of this tissue by pharmacological means have been identified. Whether or not brown adipose tissue will be useful in the battle against obesity remains to be seen. However, this possibility is certainly well worth exploring. PMID: 20386559 [PubMed - indexed for MEDLINE] | | | | | | | | | | | | | | | | | | | | | | | | | Bioresponsive dextrin-rhEGF conjugates: in vitro evaluation in models relevant to its proposed use as a treatment for chronic wounds. Mol Pharm. 2010 Jun 7;7(3):699-707 Authors: Hardwicke J, Moseley R, Stephens P, Harding K, Duncan R, Thomas DW We recently developed a bioresponsive dextrin-recombinant human epidermal growth factor (rhEGF) conjugate as a polymer therapeutic with potential for use in the promotion of tissue repair. The aim of these studies was to use patient-derived wound fluid and fibroblasts to evaluate its potential for further development as a treatment for chronic wounds, such as venous leg ulceration, a growing clinical challenge in the aging population. First, the levels of EGF (ELISA assay), alpha-amylase and elastase (enzyme assays) were measured in patient-derived acute and chronic wound fluid. EGF was detected in acute, but not in chronic wound fluid. alpha-Amylase concentrations were higher in acute (188 IU/L), compared to chronic wound fluid (52 IU/L), but both were in the range of human serum levels. Although elastase was present in chronic wound fluid (2.1 +/- 1.2 RFU/min), none was detected in acute wound fluid. Dextrin-rhEGF incubation in chronic wound fluid led to endogenous alpha-amylase-mediated release of rhEGF (ELISA) that was maximal at 48 h. When the migration of HaCaT keratinocytes and of human fibroblasts (isolated from patient-matched, normal skin and chronic dermal wounds) was studied in vitro using the scratch wound assay, enhanced cell migration was observed in response to both free rhEGF and alpha-amylase-activated dextrin-rhEGF conjugate compared to controls. In addition, fibroblasts displayed increased proliferation (normal dermal fibroblasts approximately 160%; chronic wound fibroblasts approximately 140%) following incubation (72 h) with dextrin-rhEGF that had been exposed to physiological levels of alpha-amylase (93 IU/L). These results suggest further preclinical in vivo evaluation of dextrin-rhEGF is warranted to determine whether conjugate pharmacokinetics and rhEGF liberation into such a complex and aggressive environment can still lead to bioactivity. PMID: 20166755 [PubMed - indexed for MEDLINE] | | | | | | | | | | | | | | | | | | | | | | | | | Methods to assess stem cell lineage, fate and function. Adv Drug Deliv Rev. 2010 Sep 2; Authors: Nguyen PK, Nag D, Wu JC Stem cell therapy has the potential to regenerate injured tissue. For stem cells to achieve their full therapeutic potential, stem cells must differentiate into the target cell, reach the site of injury, survive, and engraft. To fully characterize these cells, evaluation of cell morphology, lineage specific markers, cell specific function, and gene expression must be performed. To monitor survival and engraftment, cell fate imaging is vital. Only then can organ specific function be evaluated to determine the effectiveness of therapy. In this review, we will discuss methods for evaluating the function of transplanted cells for restoring the heart, nervous system, and pancreas. We will also highlight the specific challenges facing these potential therapeutic areas. PMID: 20816906 [PubMed - as supplied by publisher] | | | | | | | | | | | | | | | | | | | | | | | | | A novel controlled release drug delivery system for multiple drugs based on electrospun nanofibers containing nanoparticles. J Pharm Sci. 2010 May 3; Authors: Wang Y, Wang B, Qiao W, Yin T This study describes development of a novel controlled drug release system for multiple drugs, it consisted of Chitosan nanoparticles/PCL composite electrospun nanofibers with core-sheath structures. Two model agents' rhodamine B and naproxen were successfully loaded in the core and sheath region respectively. The behavior of these two agents demonstrated a good controlled release and temporality, providing a new way to obtain program or temporality release for multiple agents. Particularly, this is potential applications in the field of tissue engineering, sutures and wound dressings. (c) 2010 Wiley-Liss, Inc. and the American Pharmacists Association J Pharm Sci. PMID: 20821381 [PubMed - as supplied by publisher] | | | | | | | | | | | | | | | | | | | | | | | | | The Journal of Gene MedicineJapanese Society of Gene Therapy (JSGT) Young Investigator Award from 2009. J Gene Med. 2010 Sep;12(9):790-791 Authors: Kaneko S PMID: 20821749 [PubMed - as supplied by publisher] | | | | | | | | | | | | | | | | | | | | | | | | | Multiplexed, high-throughput analysis of 3D microtissue suspensions. Integr Biol (Camb). 2010 Sep 1; Authors: Chen AA, Underhill GH, Bhatia SN Three-dimensional (3D) tissue models have significantly improved our understanding of structure/function relationships and promise to lead to new advances in regenerative medicine. However, despite the expanding diversity of 3D tissue fabrication methods, approaches for functional assessment have been relatively limited. Here, we describe the fabrication of microtissue (mu-tissue) suspensions and their quantitative evaluation with techniques capable of analyzing large sample numbers and performing multiplexed parallel analysis. We applied this platform to 3D mu-tissues representing multiple stages of liver development and disease including: embryonic stem cells, bipotential hepatic progenitors, mature hepatocytes, and hepatoma cells photoencapsulated in polyethylene glycol hydrogels. Multiparametric mu-tissue cytometry enabled quantitation of fluorescent reporter expression within populations of intact mu-tissues (n>/= 10(2)-10(3)) and sorting-based enrichment of subsets for subsequent studies. Further, 3D mu-tissues could be implanted in vivo, respond to systemic stimuli, retrieved and quantitatively assessed. In order to facilitate multiplexed 'pooled' experimentation, fluorescent labeling strategies were developed and utilized to investigate the impact of mu-tissue composition and exposure to soluble factors. In particular, examination of drug/gene interactions on collections of 3D hepatoma mu-tissues indicated synergistic influence of doxorubicin and siRNA knockdown of the anti-apoptotic gene BCL-XL. Collectively, these studies highlight the broad utility of mu-tissue suspensions as an enabling approach for high n, populational analysis of 3D tissue biology in vitro and in vivo. PMID: 20820630 [PubMed - as supplied by publisher] | | | | | | | | | | | | | | | | | | | | | | | | | Elastomeric osteoconductive synthetic scaffolds with acquired osteoinductivity expedite the repair of critical femoral defects in rats. Tissue Eng Part A. 2010 Sep 6; Authors: Filion TM, Li X, Mason-Savas A, Kreider JM, Goldstein S, Ayers D, Song J Regenerative medicine aspires to reduce reliance on or overcome limitations associated with donor tissue-mediated repair. Structural bone allografts are commonly used in orthopedic surgery with a high percentage of graft failure due to poor tissue integration. This problem is aggravated among elderly, those suffering from metabolic conditions, or those undergoing cancer therapies that compromise graft healing. Towards this end, we developed a synthetic graft named FlexBone where nanocrystalline hydroxyapatite (nHA, 50 wt%) was structurally integrated with crosslinked poly(hydroxyethyl methacrylate) hydrogel that provides dimensional stability and elasticity. It recapitulates the essential role of nHA in defining the osteoconductivity and biochemical microenvironment of bone due to its affinity for biomolecules. Here we demonstrate that FlexBone effectively absorbed endogenously secreted signaling molecules associated with the inflammation/graft healing cascade upon being press-fit into a 5-mm rat femoral segmental defect. Furthermore, when preabsorbed with a single dose of 400-ng rhBMP-2/7, it enabled the functional repair of the critical-sized defect by 8-12 weeks. FlexBone was stably encapsulated by the bridging bony callus while the FlexBone-callus interface was continuously remodeled. In summary, FlexBone combines the dimensional stability and osteoconductivity of structural bone allografts with desirable surgical compressibility and acquired osteoinductivity in an easy-to-fabricate and scalable synthetic biomaterial. PMID: 20818999 [PubMed - as supplied by publisher] | | | | | | | | | | | | | | | | | | | | | | | | | Epigenetic Differences between Embryonic Stem Cells Generated from Blastocysts Developed In Vitro and In Vivo. Cell Reprogram. 2010 Sep 6; Authors: Horii T, Yanagisawa E, Kimura M, Morita S, Hatada I Abstract Embryonic stem (ES) cells constitute a very important tool for regenerative medicine today. These ES cells, and human ES cells in particular, are almost all derived from embryos obtained by in vitro fertilization (IVF) and from in vitro culture (IVC); however, such in vitro manipulated embryos often show abnormal genomic imprinting, which can lead to the development of various diseases. Nevertheless, several reports have evaluated ES cells derived from in vitro manipulated embryos. In this study, we established ES cells derived from both in vivo and in vitro developed blastocysts (Vivo ES cells and Vitro ES cells, respectively) to compare the methylation status of imprinted genes and gene expression patterns. At very early passages, Vitro ES cells showed an increase in abnormal genomic imprinting compared to Vivo ES cells. In addition, we found that the gene expression patterns of several methylation related-genes frequently shifted to promote demethylation and to inhibit methylation in early-passage Vitro ES cells. In contrast, at later passages, we found no significant differences between Vivo and Vitro ES cells. In conclusion, it is advisable to use early passage Vivo ES cells whenever feasible, or to select ES cell lines with a normal epigenotype. PMID: 20818993 [PubMed - as supplied by publisher] | | | | | | | | | | | | | | | | | | | | | | | | | Ketones and lactate "fuel" tumor growth and metastasis: Evidence that epithelial cancer cells use oxidative mitochondrial metabolism. Cell Cycle. 2010 Sep 21;9(17) Authors: Bonuccelli G, Tsirigos A, Whitaker-Menezes D, Pavlides S, Pestell RG, Chiavarina B, Frank PG, Flomenberg N, Howell A, Martinez-Outschoorn UE, Sotgia F, Lisanti MP Previously, we proposed a new model for understanding the "Warburg effect" in tumor metabolism. In this scheme, cancer-associated fibroblasts undergo aerobic glycolysis and the resulting energy-rich metabolites are then transferred to epithelial cancer cells, where they enter the TCA cycle, resulting in high ATP production via oxidative phosphorylation. We have termed this new paradigm "The Reverse Warburg Effect". Here, we directly evaluate whether the end-products of aerobic glycolysis (3-hydroxy-butyrate and L-lactate) can stimulate tumor growth and metastasis, using MDA-MB-231 breast cancer xenografts as a model system. More specifically, we show that administration of 3-hydroxy-butyrate (a ketone body) increases tumor growth by approximately 2.5-fold, without any measurable increases in tumor vascularization/ angiogenesis. Both 3-hydroxy-butyrate and L-lactate functioned as chemo-attractants, stimulating the migration of epithelial cancer cells. Although L-lactate did not increase primary tumor growth, it stimulated the formation of lung metastases by approximately 10-fold. Thus, we conclude that ketones and lactate fuel tumor growth and metastasis, providing functional evidence to support the "Reverse Warburg Effect". Moreover, we discuss the possibility that it may be unwise to use lactate-containing i.v. solutions (such as Lactated Ringer's or Hartmann's solution) in cancer patients, given the dramatic metastasis-promoting properties of L-lactate. Also, we provide evidence for the up-regulation of oxidative mitochondrial metabolism and the TCA cycle in human breast cancer cells in vivo, via an informatics analysis of the existing raw transcriptional profiles of epithelial breast cancer cells and adjacent stromal cells. Lastly, our findings may explain why diabetic patients have an increased incidence of cancer, due to increased ketone production, and a tendency towards autophagy/mitophagy in their adipose tissue. PMID: 20818174 [PubMed - as supplied by publisher] | | | | | | | | | | | | | | | | | | | | | | | | | Long-term human pluripotent stem cell self-renewal on synthetic polymer surfaces. Biomaterials. 2010 Sep 1; Authors: Brafman DA, Chang CW, Fernandez A, Willert K, Varghese S, Chien S Realization of the full potential of human pluripotent stem cells (hPSCs) in regenerative medicine requires the development of well-defined culture conditions for their long-term growth and directed differentiation. Current practices for maintaining hPSCs generally utilize empirically determined combinations of feeder cells and other animal-based products, which are expensive, difficult to isolate, subject to batch-to-batch variations, and unsuitable for cell-based therapies. Using a high-throughput screening approach, we identified several polymers that can support self-renewal of hPSCs. While most of these polymers provide support for only a short period of time, we identified a synthetic polymer poly(methyl vinyl ether-alt-maleic anhydride) (PMVE-alt-MA) that supported the long-term attachment, proliferation and self-renewal of HUES1, HUES9, and iPSCs. The hPSCs cultured on PMVE-alt-MA maintained their characteristic morphology, expressed high levels of markers of pluripotency, and retained a normal karyotype. Such cost-effective, polymer-based matrices that support long-term self-renewal and proliferation of hPSCs will not only help to accelerate the translational perspectives of hPSCs, but also provide a platform to elucidate the underlying molecular mechanisms that regulate stem cell proliferation and differentiation. PMID: 20817292 [PubMed - as supplied by publisher] | | | | | | | | | | | | | | | | | | | | | | | | | Exposure to extremely low-frequency (50 Hz) electromagnetic fields enhances adult hippocampal neurogenesis in C57BL/6 mice. Exp Neurol. 2010 Sep 2; Authors: Cuccurazzu B, Leone L, Podda MV, Piacentini R, Riccardi E, Ripoli C, Azzena GB, Grassi C Throughout life, new neurons are continuously generated in the hippocampus, which is therefore a major site of structural plasticity in the adult brain. We recently demonstrated that extremely low-frequency electromagnetic fields (ELFEFs) promote the neuronal differentiation of neural stem cells in vitro by up-regulating Ca(v)1-channel activity. The aim of the present study was to determine whether 50-Hz / 1 mT ELFEF stimulation also affects adult hippocampal neurogenesis in vivo, and if so, to identify the molecular mechanisms underlying this action and its functional impact on synaptic plasticity. ELFEF exposure (1 to 7 hours/day for 7 days) significantly enhanced neurogenesis in the dentate gyrus (DG) of adult mice, as documented by increased numbers of cells double-labeled for 5-bromo-deoxyuridine (BrdU) and doublecortin. Quantitative RT-PCR analysis of hippocampal extracts revealed significant ELFEF exposure-induced increases in the transcription of pro-neuronal genes (Mash1, NeuroD2, Hes1) and genes encoding Ca(v)1.2 channel alpha(1C) subunits. Increased expression of NeuroD1, NeuroD2 and Ca(v)1 channels was also documented by Western blot analysis. Immunofluorescence experiments showed that, 30 days after ELFEF stimulation, roughly half of the newly generated immature neurons had survived and become mature dentate granule cells (as shown by their immunoreactivity for both BrdU and NeuN) and were integrated into the granule cell layer of the DG. Electrophysiological experiments demonstrated that the new mature neurons influenced hippocampal synaptic plasticity, as reflected by increased long-term potentiation. Our findings show that ELFEF exposure can be an effective tool for increasing in vivo neurogenesis, and they could lead to the development of novel therapeutic approaches in regenerative medicine. PMID: 20816824 [PubMed - as supplied by publisher] | | | | | | | | | | | | | | | | | | | | | | | | | Neural development of methyl-CpG-binding protein 2 null embryonic stem cells: A system for studying Rett syndrome. Brain Res. 2010 Sep 2; Authors: Okabe Y, Kusaga A, Takahashi T, Mitsumasu C, Murai Y, Tanaka E, Higashi H, Matsuishi T, Kosai KI Mutations in methyl-CpG-binding protein 2 (MeCP2) gene cause the neurodevelopmental disorder Rett syndrome (RTT). Here, we describe a new experimental system that efficiently elucidates the role of MeCP2 in neural development. MeCP2-null and control ES cells were generated by adenoviral conditional targeting and examined for maintenance of the undifferentiated ES cell state, neurogenesis, and gliogenesis during in vitro differentiation. In addition, dopamine release and electrophysiological features of neurons differentiated from these ES cells were examined. Loss of MeCP2 did not affect undifferentiated ES cell colony morphology and growth, or the timing or efficiency of neural stem cell differentiation into Nestin-, TuJ- or TH-positive neurons. In contrast, gliogenesis was drastically accelerated by MeCP2 deficiency. Dopamine production and release in response to a depolarizing stimulus in MeCP2-null ES-derived dopaminergic neurons was intact. However, MeCP2-null differentiated neurons showed significantly smaller voltage-dependent Na(+) currents and A-type K(+) currents, suggesting incomplete maturation. Thus, MeCP2 is not essential for maintenance of the undifferentiated ES cell state, neurogenesis, or dopaminergic function; rather, it is principally involved in inhibiting gliogenesis. Altered neuronal maturity may indirectly result from abnormal glial development and may underlie the pathogenesis of RTT. These data contribute to a better understanding of the developmental roles of MeCP2 and the pathogenesis of RTT. PMID: 20816763 [PubMed - as supplied by publisher] | | | | | | | | | | | | | | | | | | | | | | | | | Nanotopography/Mechanical Induction of Stem-Cell Differentiation. Methods Cell Biol. 2010;98C:241-294 Authors: Teo BK, Ankam S, Chan LY, Yim EK The interplay of biophysical and biochemical cues in the extracellular microenvironment regulate and control the cell fate of stem cells. Understanding the interaction between stem cells and the extracellular substrate will be crucial in controlling stem cell differentiation for regenerative medicine applications. One of the biophysical properties of the microenvironment is substrate topology, which has been demonstrated to be an important mediator of stem cell lineage regulation. Biomimetic microenvironment topology can be engineered by chemical patterning or physical patterning. The rapid advancements in nanofabrication techniques have enabled versatility in patterning types with controlled chemistries, geometries and sizes. The chapter will focus on discussing the effect on physical nanotopography on stem cell differentiation and the current theories on the topography/ mechanical force induction of stem cell differentiation possibly through integrin clustering, focal adhesion, cytoskeleton organization and the nuclear mechanosensing to sense and integrate these biophysical signals from the extracellular microenvironment. PMID: 20816238 [PubMed - as supplied by publisher] | | | | | | | | | | | | | | | | | | | | | | | | | Generation of rat pancreas in mouse by interspecific blastocyst injection of pluripotent stem cells. Cell. 2010 Sep 3;142(5):787-99 Authors: Kobayashi T, Yamaguchi T, Hamanaka S, Kato-Itoh M, Yamazaki Y, Ibata M, Sato H, Lee YS, Usui J, Knisely AS, Hirabayashi M, Nakauchi H The complexity of organogenesis hinders in vitro generation of organs derived from a patient's pluripotent stem cells (PSCs), an ultimate goal of regenerative medicine. Mouse wild-type PSCs injected into Pdx1(-/-) (pancreatogenesis-disabled) mouse blastocysts developmentally compensated vacancy of the pancreatic "developmental niche," generating almost entirely PSC-derived pancreas. To examine the potential for xenogenic approaches in blastocyst complementation, we injected mouse or rat PSCs into rat or mouse blastocysts, respectively, generating interspecific chimeras and thus confirming that PSCs can contribute to xenogenic development between mouse and rat. The development of these mouse/rat chimeras was primarily influenced by host blastocyst and/or foster mother, evident by body size and species-specific organogenesis. We further injected rat wild-type PSCs into Pdx1(-/-) mouse blastocysts, generating normally functioning rat pancreas in Pdx1(-/-) mice. These data constitute proof of principle for interspecific blastocyst complementation and for generation in vivo of organs derived from donor PSCs using a xenogenic environment. PMID: 20813264 [PubMed - in process] | | | | | | | | | | | | | | | | | | | | | | | | | [Interventional, intramyocardial stem cell therapy in ischemic cardiomyopathy: update 2010.] Herz. 2010 Jul 29; Authors: Bergmann MW, Jaquet K, Schneider C, Krause K, Ujeyl A, Kuck KH BACKGROUND: The intracoronary application of autologous bone marrow cells has proven hitherto to be safe but not sufficiently effective in patients with ischemic cardiomyopathy. The interventional application of cells injected directly into the myocardium represents one possible approach to improve effectiveness. TECHNIQUES: The NOGA method is based on the CARTO technique, which has been evaluated extensively for safety and feasibility in patients with heart failure. In a first step, an electrically and anatomically exact map of the left ventricle is obtained. Guided by this three-dimensional map direct injection of the cells into the ischemic area can be easily performed. CLINICAL STUDIES: Since its introduction in 2002 many studies have proven the safety, feasibility and effectiveness of NOGA-guided regenerative therapy to the left ventricle. While several studies also suggest effectiveness regarding various parameters of left ventricular function, no larger multicenter study is available to date. Such studies with also clinical endpoints are currently ongoing. CONCLUSION: The currently available data support, but do not yet prove, the hypothesis that intramyocardial stem cell therapy using NOGA-guided injection into the myocardium is safe and feasible in both acute and chronic ischemic cardiomyopathy. Ongoing trials will reveal whether this approach will become the standard form for applying cell therapy to the heart. PMID: 20814657 [PubMed - as supplied by publisher] | | | | | | | | | |  | |  | |  |
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