|  |  |  | | | | | TE-RegenMed-StemCell feed | | | | | | | | | | | | | | | | | | | Allogeneic periodontal ligament stem cell therapy for periodontitis in Swine. Stem Cells. 2010 Oct;28(10):1829-38 Authors: Ding G, Liu Y, Wang W, Wei F, Liu D, Fan Z, An Y, Zhang C, Wang S Periodontitis is one of the most widespread infectious diseases in humans. It is the main cause of tooth loss and associated with a number of systemic diseases. Until now, there is no appropriate method for functional periodontal tissue regeneration. Here, we establish a novel approach of using allogeneic periodontal ligament stem cells (PDLSCs) sheet to curing periodontitis in a miniature pig periodontitis model. Significant periodontal tissue regeneration was achieved in both the autologous and the allogeneic PDLSCs transplantation group at 12 weeks post-PDLSCs transplantation. Based on clinical assessments, computed tomography (CT) scanning, and histological examination, there was no marked difference between the autologous and allogeneic PDLSCs transplantation groups. In addition, lack of immunological rejections in the animals that received the allogeneic PDLSCs transplantation was observed. Interestingly, we found that human PDLSCs fail to express human leukocyte antigen (HLA)-II DR and costimulatory molecules. PDLSCs were not able to elicit T-cell proliferation and inhibit T-cell proliferation when stimulated with mismatched major histocompatibility complex molecules. Furthermore, we found that prostaglandin E2 (PGE2) plays a crucial role in PDLSCs-mediated immunomodulation and periodontal tissue regeneration in vitro and in vivo. Our study demonstrated that PDLSCs possess low immunogenicity and marked immunosuppression via PGE2-induced T-cell anergy. We developed a standard technological procedure of using allogeneic PDLSCs to cure periodontitis in swine. STEM CELLS 2010;28:1829-1838. PMID: 20979138 [PubMed - in process] | | | | | | | | | | | | | | | | | | | | | | | | | Properties of hepatocyte-like cell clusters from human adipose tissue-derived mesenchymal stem cells. Tissue Eng Part C Methods. 2010 Aug;16(4):761-70 Authors: Okura H, Komoda H, Saga A, Kakuta-Yamamoto A, Hamada Y, Fumimoto Y, Lee CM, Ichinose A, Sawa Y, Matsuyama A There are only a few reports that describe the hepatocytic differentiation potential of human adipose tissue-derived mesenchymal stem cells (hADMSCs) and no reports that describe the in vivo functions of hepatocyte-like cells differentiated from somatic stem cells including hADMSCs. In this study, we established a new method for generation of functional hepatocyte-like cell clusters using floating culture method and induced functional hepatocyte-like cell clusters, which functioned effectively not only in vitro but also in vivo. The generated hepatocyte-like cell clusters were characterized by gene expression analysis, functional assays, and transplantation into non-obese diabetic severe combined immunodeficiency (NOD-SCID) mouse with chronic liver injury. The generated hepatocyte-like cell clusters expressed various genes normally found on mature hepatocytes. The cell clusters exhibited functional characteristics of hepatocytes: they expressed albumin, secreted urea, had cytochrome P450 activity, could take up low-density lipoprotein, and stored glycogen. Transplantation of these cell clusters into NOD-SCID mouse with chronic liver injury resulted in a significant improvement of serum albumin and total bilirubin levels. In summary, we established a new protocol for efficient induction of hADMSCs into functional hepatocyte-like cell clusters. PMID: 19839740 [PubMed - indexed for MEDLINE] | | | | | | | | | | | | | | | | | | | | | | | | | A comparison of imaging methodologies for 3D tissue engineering. Microsc Res Tech. 2010 Dec;73(12):1123-33 Authors: Smith LE, Smallwood R, Macneil S Imaging of cells in two dimensions is routinely performed within cell biology and tissue engineering laboratories. When biology moves into three dimensions imaging becomes more challenging, especially when multiple cell types are used. This review compares imaging techniques used regularly in our laboratory in the culture of cells in both two and three dimensions. The techniques reviewed include phase contrast microscopy, fluorescent microscopy, confocal laser scanning microscopy, electron microscopy, and optical coherence tomography. We compare these techniques to the current "gold standard" for imaging three-dimensional tissue engineered constructs, histology. Microsc. Res. Tech. 73:1123-1133, 2010. © 2010 Wiley-Liss, Inc. PMID: 20981758 [PubMed - in process] | | | | | | | | | | | | | | | | | | | | | | | | | Preparation, characterization and in vitro testing of poly(lactic-co-glycolic) acid/barium titanate nanoparticle composites for enhanced cellular proliferation. Biomed Microdevices. 2010 Oct 28; Authors: Ciofani G, Ricotti L, Mattoli V The recent advancements in tissue engineering and, more in general, in cell-based applications, has led to an ever increasing interest toward new materials for sustained cell proliferation and differentiation. Here, the preparation and the characterization of scaffolds based on poly(lactic-co-glycolic) acid / barium titanate nanoparticle composites are presented. In vitro testing on H9C2 cell line demonstrates how the presence of the nanoparticles positively affects both the proliferation and the differentiation of this muscle-like cell line. Finally, the possibility to obtain porous scaffold and, therefore, an actual 3D culture system, is introduced. PMID: 20981490 [PubMed - as supplied by publisher] | | | | | | | | | | | | | | | | | | | | | | | | | Evaluating cell proliferation based on internal pore size and 3D scaffold architecture fabricated using solid freeform fabrication technology. J Mater Sci Mater Med. 2010 Oct 28; Authors: Lee JW, Ahn G, Kim JY, Cho DW The scaffold, as a medical component to regenerate tissues or organs in humans, plays an important role in tissue engineering. Recently, solid freeform fabrication (SFF) technology using computer-assisted methods was applied to address the problems of conventional fabrication methods in which the internal/outer architectures cannot be controlled. In this report, we propose suitable scaffolds for bone tissue regeneration considering the internal pore size and scaffold architecture. Poly(propylene fumarate) was used as the biodegradable photopolymer, and scaffolds were fabricated using microstereolithography (MSTL). We observed the relationship between the internal pores and architecture, and the proliferation of pre-osteoblast cells. To demonstrate the superiority of MSTL, we fabricated conventional and SFF scaffolds, and measured the cell proliferation rates for each. The results showed that cell proliferation on the MSTL scaffold was clearly superior and indicated that MSTL would be a good replacement for current conventional methods. PMID: 20981473 [PubMed - as supplied by publisher] | | | | | | | | | | | | | | | | | | | | | | | | | Surface lipids as multifunctional mediators of skin responses to environmental stimuli. Mediators Inflamm. 2010;2010:321494 Authors: De Luca C, Valacchi G Skin surface lipid (SSL) film is a mixture of sebum and keratinocyte membrane lipids, protecting skin from environment. Its composition is unique for the high percentage of long chain fatty acids, and of the polyterpenoid squalene, absent in other human tissues, and in non-human Primates sebum. Here, the still incomplete body of information on SSL as mediators of external chemical, physical, and microbial signals and stressors is revised, focusing on the central event of the continuous oxidative modification induced by the metabolic activity of residential and pathological microbial flora, natural or iatrogenic UV irradiation, exposure to chemicals and cosmetics. Once alpha-tocopherol and ubiquinol-10 antioxidant defences of SSL are overcome, oxidation of squalene and cholesterol gives rise to reactive by-products penetrating deeper into skin layers, to mediate local defensive inflammatory, photo-protective, immune reactions or, at higher concentrations, inducing local but also systemic immune depression, ultimately implicating skin cancerogenesis. Qualitative modifications of SSL represent a pathogenetic sign of diagnostic value in dermatological disorders involving altered sebum production, like pytiriasis versicolor, acne, atopic or seborrheic dermatitis, as well as photo-aging. Achievements of nutriceutical interventions aimed at restoring normal SSL composition and homeostasis are discussed, as feasible therapeutic goals and major means of photo-protection. PMID: 20981292 [PubMed - in process] | | | | | | | | | | | | | | | | | | | | | | | | | The Role of the IL-12 Cytokine Family in Directing T-Cell Responses in Oral Candidosis. Clin Dev Immunol. 2011;2011:697340 Authors: Wei XQ, Rogers H, Lewis MA, Williams DW Candida albicans is an opportunistic fungal pathogen that normally exists as a harmless commensal in humans. In instances where host debilitation occurs, Candida can cause a range of clinical infections, and whilst these are primarily superficial, effecting mucosal membranes, systemic infections can develop in severely immunocompromised individuals. The mechanism of host immunity during commensal carriage of C. albicans has been intensively studied. In this paper, we present the most recent information concerning host recognition of C. albicans leading to cytokine production and the subsequent T-cell responses generated in response to C. albicans. Particular focus is given to the role of the IL-12 cytokine family including IL-12, IL-23, IL-27, and IL-35, in host immunity to Candida. CD4(+) T-cells are considered crucial in the regulation of immunity and inflammation. In this regard, the role of Th1/2, helper cells, together with the recently identified Th17 and Treg cells in candidosis will be discussed. Understanding the detailed mechanisms that underlie host immunity to Candida not only will be of benefit in terms of the infections caused by this organism but could also be exploited in the development of therapeutic interventions for other diseases. PMID: 20981280 [PubMed - in process] | | | | | | | | | | | | | | | | | | | | | | | | | Thermoresponsive poly(N-isopropyl acrylamide)-grafted polycaprolactone films with surface immobilization of collagen. Colloids Surf B Biointerfaces. 2010 Oct 8; Authors: Xu FJ, Zheng YQ, Zhen WJ, Yang WT Thermoresponsive poly(N-isopropylacrylamide) (P(NIPAAm))-grafted polycaprolactone (PCL) films with a suitable amount of immobilized cell-adhesive collagen were prepared to improve cell adhesion and proliferation above the lower critical solution temperature (LCST, 32°C) of P(NIPAAm) without destroying cell detachment properties at lower temperatures. Covalently tethered P(NIPAAm) brushes on PCL film surfaces were first prepared via surface-initiated atom transfer radical polymerization (ATRP). The alkyl bromide end groups of the grafted P(NIPAAm) brushes were used in nucleophilic substitution reactions for the direct coupling of collagen to produce the collagen-immobilized thermoresponsive PCL surface. At 37°C, the cell attachments on the collagen-immobilized thermoresponsive PCL surface were enhanced substantially. The attached cells could be recovered simply by lowering culture temperature. The P(NIPAAm)-grafted PCL films with immobilized collagen are potentially useful as adhesion modifiers for advanced cell culture and tissue engineering applications. PMID: 20980132 [PubMed - as supplied by publisher] | | | | | | | | | | | | | | | | | | | | | | | | | Biocompatibility and osteogenesis of biomimetic Bioglass-Collagen-Phosphatidylserine composite scaffolds for bone tissue engineering. Biomaterials. 2010 Oct 25; Authors: Xu C, Su P, Chen X, Meng Y, Yu W, Xiang AP, Wang Y A novel biomimetic composite scaffold Bioglass-Collagen-Phosphatidylserine (BG-COL-PS) was fabricated with a freeze-drying technique. The macrostructure and morphology as well as mechanical strength of the scaffolds were characterized. Scanning electronic microscopy (SEM) showed that the BG-COL-PS scaffolds exhibited interconnected porous structures with pore sizes of several microns up to about 300 μm. The scaffolds have a porosity of 75.40% and the corresponding compressive strength of 1.5469 Mpa. Rat mesenchymal stem cells (rMSCs) were seeded on BG-COL-PS or BG-COL scaffolds and cultured for 21 days in vitro. Based on the results of SEM, dsDNA content, alkaline phosphatase (ALP) activity, osteogenic gene expression analysis and alizarin red staining, the responses of MSCs to the scaffold exhibited a higher degree of attachment, growth as well as osteogenic differentiation than those on BG-COL scaffolds in vitro. To investigate the in vivo biocompatibility and osteogenesis of the composite scaffolds, both pure BG-COL-PS scaffolds and MSC/scaffold constructs were implanted in rat femurs defects for 6 weeks and studied histologically and radiographically. The in vivo results showed that BG-COL-PS composite scaffolds exhibited good biocompatibility and extensive osteoconductivity with host bone. Moreover, the BG-COL-PS/MSC constructs dramatically enhanced the efficiency of new bone formation than pure BG-COL-PS scaffolds or BG-COL/MSC constructs. All these results demonstrate the usefulness of PS composited BG-COL-PS scaffolds for inducing enhanced bone formation. The BG-COL-PS scaffolds fulfill the basic requirements of bone tissue engineering scaffold and have the potential to be applied in orthopedic and reconstructive surgery. PMID: 20980051 [PubMed - as supplied by publisher] | | | | | | | | | | | | | | | | | | | | | | | | | Chitosan/Poly(ɛ-caprolactone) blend scaffolds for cartilage repair. Biomaterials. 2010 Oct 25; Authors: Neves SC, Moreira Teixeira LS, Moroni L, Reis RL, Van Blitterswijk CA, Alves NM, Karperien M, Mano JF Chitosan (CHT)/poly(ɛ-caprolactone) (PCL) blend 3D fiber-mesh scaffolds were studied as possible support structures for articular cartilage tissue (ACT) repair. Micro-fibers were obtained by wet-spinning of three different polymeric solutions: 100:0 (100CHT), 75:25 (75CHT) and 50:50 (50CHT) wt.% CHT/PCL, using a common solvent solution of 100 vol.% of formic acid. Scanning electron microscopy (SEM) analysis showed a homogeneous surface distribution of PCL. PCL was well dispersed throughout the CHT phase as analyzed by differential scanning calorimetry and Fourier transform infrared spectroscopy. The fibers were folded into cylindrical moulds and underwent a thermal treatment to obtain the scaffolds. μCT analysis revealed an adequate porosity, pore size and interconnectivity for tissue engineering applications. The PCL component led to a higher fiber surface roughness, decreased the scaffolds swelling ratio and increased their compressive mechanical properties. Biological assays were performed after culturing bovine articular chondrocytes up to 21 days. SEM analysis, live-dead and metabolic activity assays showed that cells attached, proliferated, and were metabolically active over all scaffolds formulations. Cartilaginous extracellular matrix (ECM) formation was observed in all formulations. The 75CHT scaffolds supported the most neo-cartilage formation, as demonstrated by an increase in glycosaminoglycan production. In contrast to 100CHT scaffolds, ECM was homogenously deposited on the 75CHT and 50CHT scaffolds. Although mechanical properties of the 50CHT scaffold were better, the 75CHT scaffold facilitated better neo-cartilage formation. PMID: 20980050 [PubMed - as supplied by publisher] | | | | | | | | | | | | | | | | | | | | | | | | | [Tissue engineering vascularized bone repairing segmental femoral bone defects in rabbits.] Zhonghua Yi Xue Za Zhi. 2010 Jun;90(23):1637-1641 Authors: Wang L, Pei GX, Gao LB, Jiang S, Mu TW, Chen SY, Qin JJ, Jin D, Lou AJ, Zhao PR OBJECTIVE: To investigate the effectiveness and mechanism of tissue engineering vascularized bone in repairing segmental femoral bone defects in rabbits. METHODS: Thirty-two rabbits were randomized into two groups (n = 16 each). A segmental and critical bone defect of 15 mm in length was made at left femur. In experimental group, the tissue engineering bone constructed from autologous bone marrow mesenchymal stem cells plus β-tricalcium phosphate (β-TCP) and vascular bundle was implanted into bony defect. In control group, there was no implantation of vascular bundle. Animals were sacrificed at 2, 4, 8 and 12 weeks post-implantation respectively. Histological observation was conducted to determine the process of new bone formation and remolding. The expression of vascular endothelial growth factor (VEGF) in new bone was measured by immunohistochemistry, real-time PCR and Western blot. RESULTS: As indicated by histological observations over time, new bone formation increased in both groups. It was better in the experimental group than the control group at the beginning of 4 weeks. The expression level of VEGF gradually decreased in each group after an initial rise. And the expression of VEGF was significantly higher than the control group after implantation at all time points and peaked at 4 weeks. CONCLUSION: Tissue engineering vascularized bone accelerates bone repair in critical size defect model of femur in rabbit. Implantation of vascular bundle can promote the secretion of VEGF. And VEGF is an essential mediator of both angiogenesis and ossification. PMID: 20979756 [PubMed - as supplied by publisher] | | | | | | | | | | | | | | | | | | | | | | | | | Behaviors of NIH-3T3 Fibroblasts on Graphene/Carbon Nanotubes: Proliferation, Focal Adhesion, and Gene Transfection Studies. ACS Nano. 2010 Oct 27; Authors: Ryoo SR, Kim YK, Kim MH, Min DH Carbon-based materials, including graphene and carbon nanotubes, have been considered attractive candidates for biomedical applications such as scaffolds in tissue engineering, substrates for stem cell differentiation, and components of implant devices. Despite the potential biomedical applications of these materials, only limited information is available regarding the cellular events, including cell viability, adhesion, and spreading, that occur when mammalian cells interface with carbon-based nanomaterials. Here, we report behaviors of mammalian cells, specifically NIH-3T3 fibroblast cells, grown on supported thin films of graphene and carbon nanotubes to investigate biocompatibility of the artificial surface. Proliferation assay, cell shape analysis, focal adhesion study, and quantitative measurements of cell adhesion-related gene expression levels by RT-PCR reveal that the fibroblast cells grow well, with different numbers and sizes of focal adhesions, on graphene- and carbon nanotube-coated substrates. Interestingly, the gene transfection efficiency of cells grown on the substrates was improved up to 250% that of cells grown on a cover glass. The present study suggests that these nanomaterials hold high potential for bioapplications showing high biocompatibility, especially as surface coating materials for implants, without inducing notable deleterious effects while enhancing some cellular functions (i.e., gene transfection and expression). PMID: 20979372 [PubMed - as supplied by publisher] | | | | | | | | | | | | | | | | | | | | | | | | | Cell line models for differentiation: preadipocytes and adipocytes. Exp Biol Med (Maywood). 2010 Oct 1;235(10):1185-93 Authors: Poulos SP, Dodson MV, Hausman GJ In vitro models have been invaluable in determining the mechanisms involved in adipocyte proliferation, differentiation, adipokine secretion and gene/protein expression. The cells presently available for research purposes all have unique advantages and disadvantages that one should be aware of when selecting cells. Established cell lines, such as 3T3-L1 cells, are easier and less costly to use than freshly isolated cells, even though freshly isolated cells allow for various comparisons such as the in vitro evaluation of different in vivo conditions that may not be possible using cell lines. Moreover, stem cells, transdifferentiated cells or dedifferentiated cells are relatively new cell models being evaluated for the study of adipocyte regulation and physiology. The focus of this brief review is to highlight similarities and differences in adipocyte models to aid in appropriate model selection and data interpretation for successful advancement of our understanding of adipocyte biology. PMID: 20864461 [PubMed - indexed for MEDLINE] | | | | | | | | | | | | | | | | | | | | | | | | | Bioengineered vascular graft grown in the mouse peritoneal cavity. J Vasc Surg. 2010 Oct;52(4):994-1002, 1002.e1-2 Authors: Song L, Wang L, Shah PK, Chaux A, Sharifi BG BACKGROUND: We tested the hypothesis that the mouse peritoneum can function like a bioreactor to generate directed bio-engineered tissues such as those used for bypass grafting. Additionally, we reasoned that the mouse animal model would allow us to elucidate the underlying cellular and molecular mechanisms that are responsible for the generation of tissue in peritoneal cavity. METHODS: Plastic tubes (two tubes/mouse) were implanted into the peritoneal cavity of three strains of mice (C57BL/6, BALB/c, and MRL). The tubes were harvested, tissue capsule surrounding the tubes was removed, and analyzed by immunostaining (five capsules/five mice/strain) and microarray (three capsules/three mice/strain). In addition, the tissue capsules that were harvested from MRL mice (n = 21) were grafted into abdominal aorta of the same mice as autografts. The patency of all grafts was monitored by micro-ultrasound, and their functionality was assessed by laser Doppler imaging of blood flow in femoral arteries. Venous (n = 13) and arterial isografts (n = 11) were used as positive controls. In a negative control group (five mice/strain), the abdominal aorta was occluded by double ligation with 9-0 silk. RESULTS: The implanted plastic tubes required at least 8 weeks of incubation in the peritoneum of the three strains of mice in order to generate useful grafts. No vascular cells were found in the tissue capsules. Microarray analysis of tissue capsules revealed that the capsular cells express a gene expression program that is vastly shared among the three strains of mice, and the cells exhibit a high degree of plasticity. The micro-ultrasound analysis of the grafts showed that 62% of autografts remained patent compared with 77% of venous isografts and 91% of arterial isografts. The laser Doppler imaging analysis showed that blood flow dropped by 40% and 35% in the autografts and vein isografts, respectively, 1 day after surgery. The flow, however, rebounded to the level of arterial isografts 1 month post-surgery and remained unchanged among all grafts for the next 4 months. Immunostaining of the autografts showed a thick vessel wall with endothelial cells that lined the lumen and smooth muscle cells that constituted the graft wall. CONCLUSION: The mouse peritoneal cavity of mice has the ability to function like a bioreactor to generate bio-engineered tissues. The tissue capsules harvested from peritoneal cavity of a mouse are composed of nonvascular cells that display phenotype of progenitor cells. After grafting, however, the capsule autografts become arterialized and remained patent for at least 4 months after surgery, similar to venous or arterial isografts. PMID: 20692791 [PubMed - indexed for MEDLINE] | | | | | | | | | | | | | | | | | | | | | | | | | Update in cartilage bio-engineering. Joint Bone Spine. 2010 Jul;77(4):283-6 Authors: Gabay O, Sanchez C, Taboas JM PMID: 20478735 [PubMed - indexed for MEDLINE] | | | | | | | | | | | | | | | | | | | | | | | | | Paraffin embedding allows effective analysis of proliferation, survival, and immunophenotyping of cells cultured on poly(l-lactic acid) electrospun nanofiber scaffolds. Tissue Eng Part C Methods. 2010 Aug;16(4):751-60 Authors: Foroni L, Dirani G, Gualandi C, Focarete ML, Pasquinelli G Morphological and immunophenotypic characterization of cells grown on poly(l-lactic acid) (PLLA) electrospun scaffolds is usually performed using immunofluorescence and cryosections. However, these methods present practical limits; histological processing, on the other hand, is believed to lead to artifactual changes in the scaffold structure. Here the formalin-fixed paraffin-embedding (FFPE) procedure was tailored to process PLLA electrospun scaffolds grown with human umbilical vein endothelial cells. After 1 to 7 days of culture, the scaffolds were processed with the FFPE procedure. Using this protocol, not only cross sections but also "en face" sections were obtained. This made possible to perform the effective light microscopy analysis of cell morphology and to assess cell adhesion and penetration without considerable scaffold damage. The method was also suitable for immunohistochemical assays, such as proliferation (Ki67), extracellular matrix production (type IV collagen), survival (cleaved caspase-3), and immunophenotyping (KDR, CD44, vimentin, CD45); results were compared with those obtained using complementary techniques (scanning electron microscopy, Alamar Blue assay, and cryosections). The FFPE protocol can be safely applied to PLLA scaffolds and provides information that are essential to study the mechanisms of interaction between cells and PLLA fibers before their potential implantation in vivo. PMID: 19824801 [PubMed - indexed for MEDLINE] | | | | | | | | | | | | | | | | | | | | | | | | | A novel bioreactor for stimulating skeletal muscle in vitro. Tissue Eng Part C Methods. 2010 Aug;16(4):711-8 Authors: Donnelly K, Khodabukus A, Philp A, Deldicque L, Dennis RG, Baar K For over 300 years, scientists have understood that stimulation, in the form of an electrical impulse, is required for normal muscle function. More recently, the role of specific parameters of the electrical impulse (i.e., the pulse amplitude, pulse width, and work-to-rest ratio) has become better appreciated. However, most existing bioreactor systems do not permit sufficient control over these parameters. Therefore, the aim of the current study was to engineer an inexpensive muscle electrical stimulation bioreactor to apply physiologically relevant electrical stimulation patterns to tissue-engineered muscles and monolayers in culture. A low-powered microcontroller and a DC-DC converter were used to power a pulse circuit that converted a 4.5 V input to outputs of up to 50 V, with pulse widths from 0.05 to 4 ms, and frequencies up to 100 Hz (with certain operational limitations). When two-dimensional cultures were stimulated at high frequencies (100 Hz), this resulted in an increase in the rate of protein synthesis (at 12 h, control [CTL] = 5.0 + or - 0.16; 10 Hz = 5.0 + or - 0.07; and 100 Hz = 5.5 + or - 0.13 fmol/min/mg) showing that this was an anabolic signal. When three-dimensional engineered muscles were stimulated at 0.1 ms and one or two times rheobase, stimulation improved force production (CTL = 0.07 + or - 0.009; 1.25 V/mm = 0.10 + or - 0.011; 2.5 V/mm = 0.14146 + or - 0.012; and 5 V/mm = 0.03756 + or - 0.008 kN/mm(2)) and excitability (CTL = 0.53 + or - 0.022; 1.25 V/mm = 0.44 + or - 0.025; 2.5 V/mm = 0.41 + or - 0.012; and 5 V/mm = 0.60 + or - 0.021 V/mm), suggesting enhanced maturation. Together, these data show that the physiology and function of muscles can be improved in vitro using a bioreactor that allows the control of pulse amplitude, pulse width, pulse frequency, and work-to-rest ratio. PMID: 19807268 [PubMed - indexed for MEDLINE] | | | | | | | | | | | | | | | | | | | | | | | | | Intravital molecular imaging of small-diameter tissue-engineered vascular grafts in mice: a feasibility study. Tissue Eng Part C Methods. 2010 Aug;16(4):597-607 Authors: Hjortnaes J, Gottlieb D, Figueiredo JL, Melero-Martin J, Kohler RH, Bischoff J, Weissleder R, Mayer JE, Aikawa E OBJECTIVES: Creating functional small-diameter tissue-engineered blood vessels has not been successful to date. Moreover, the processes underlying the in vivo remodeling of these grafts and the fate of cells seeded onto scaffolds remain unclear. Here we addressed these unmet scientific needs by using intravital molecular imaging to monitor the development of tissue-engineered vascular grafts (TEVG) implanted in mouse carotid artery. METHODS AND RESULTS: Green fluorescent protein-labeled human bone marrow-derived mesenchymal stem cells and cord blood-derived endothelial progenitor cells were seeded on polyglycolic acid-poly-L-lactic acid scaffolds to construct small-caliber TEVG that were subsequently implanted in the carotid artery position of nude mice (n = 9). Mice were injected with near-infrared agents and imaged using intravital fluorescence microscope at 0, 7, and 35 days to validate in vivo the TEVG remodeling capability (Prosense680; VisEn, Woburn, MA) and patency (Angiosense750; VisEn). Imaging coregistered strong proteolytic activity and blood flow through anastomoses at both 7 and 35 days postimplantation. In addition, image analyses showed green fluorescent protein signal produced from mesenchymal stem cell up to 35 days postimplantation. Comprehensive correlative histopathological analyses corroborated intravital imaging findings. CONCLUSIONS: Multispectral imaging offers simultaneous characterization of in vivo remodeling enzyme activity, functionality, and cell fate of viable small-caliber TEVG. PMID: 19751103 [PubMed - indexed for MEDLINE] | | | | | | | | | | | | | | | | | | | | | | | | | Validation system of tissue-engineered epithelial cell sheets for corneal regenerative medicine. Tissue Eng Part C Methods. 2010 Aug;16(4):553-60 Authors: Hayashi R, Yamato M, Takayanagi H, Oie Y, Kubota A, Hori Y, Okano T, Nishida K Recently, regenerative therapy with tissue-engineered epithelial cell sheets has been performed for treating ocular surface disease. It would be required to develop the validation method for these cell sheets to standardize and spread the regenerative therapy. In the present study, we developed a validation system for cultivated epithelial cell sheets. Human limbal epithelial cells and human oral mucosal epithelial cells were cultured with 3T3 feeder layer cells on temperature-responsive culture inserts for three different culture periods, and subjected to cell sheet harvest and validation. Epithelial cells cultured for a short period were not successfully harvested as intact contiguous cell sheets. On the other hand, total cell number and viability of epithelial cell sheets harvested after prolonged culture period decreased. Further, these cells also lost epithelial barrier function. These results showed the potential effectiveness of the proposed validation system that can evaluate fabricated cell sheets before transplantation. PMID: 19722828 [PubMed - indexed for MEDLINE] | | | | | | | | | | | | | | | | | | | | | | | | | Regression and persistence: remodelling in a tissue engineered axial vascular assembly. J Cell Mol Med. 2009 Oct;13(10):4166-75 Authors: Polykandriotis E, Euler S, Arkudas A, Pryymachuk G, Beier JP, Greil P, Dragu A, Lametschwandtner A, Kneser U, Horch RE In later stages of vasculoangiogenesis a vascular network is going through a metamorphosis for optimal perfusion and economy of energy. In this study we make a quantitative approach to phenomena of remodelling in a bioartificial neovascular network and suggest variance of calibre as a parameter of neovascular maturation. For this study, 18 male Lewis rats were subjected to the AV loop operation in combination with a hard porous biogenic matrix and an isolation chamber. The animals were allocated into three groups for different explantation intervals set to 2, 4 and 8 weeks, respectively. Collective attributes like vascular density, percent fractional area and variance of calibre were evaluated for a predefined region of interest (ROI). Late morphogenesis was evaluated by means of scanning electron microscopy. After the fourth week the absolute number of vessels within the ROI decreased (P < 0.03) whereas, on the contrary, the fractional area of all segments increased (P < 0.02). The variance in calibre was significantly increased in the 8-week group (P < 0.05). Lymphatic growth after week 4, early pericyte migration as well as intussusceptive angiogenesis were identified immunohistologically. Phenomena of remodelling were evaluated quantitatively in a neovascular network and variance could be proposed as a parameter of net vascular maturation. PMID: 19555425 [PubMed - indexed for MEDLINE] | | | | | | | | | | | | | | | | | | | | | | | | | How reproductive and regenerative medicine meet in a Chinese fertility clinic. Interviews with women about the donation of embryos to stem cell research. J Med Ethics. 2010 Oct 27; Authors: Mitzkat A, Haimes E, Rehmann-Sutter C The social interface between reproductive medicine and embryonic stem cell research has been investigated in a pilot study at a large IVF clinic in central China. Methods included observation, interviews with hospital personnel, and five in-depth qualitative interviews with women who underwent IVF and who were asked for their consent to the donation of embryos for use in medical (in fact human embryonic stem cell) research. This paper reports, and discusses from an ethical perspective, the results of an analysis of these interviews. The participants talked of extreme social pressure to become pregnant. Once they had a baby, 'spare' embryos lost practical significance due to the Chinese one-child policy. In the context of decision making about donating embryos to research, the women used the clinical distinctions between 'good and bad quality' embryos and also between frozen and transferred embryos, as guiding moral distinctions. In the absence of concrete information about what sort of research their embryos should be used for, the women interviewed either refused consent (for fear that the embryo would be given to another couple) or accepted, expressing motives of solidarity with other women in a similar situation. This reveals that they filled the knowledge gap with an image of research improving fertility treatment. PMID: 20980331 [PubMed - as supplied by publisher] | | | | | | | | | | | | | | | | | | | | | | | | | Chitosan/Poly(ɛ-caprolactone) blend scaffolds for cartilage repair. Biomaterials. 2010 Oct 25; Authors: Neves SC, Moreira Teixeira LS, Moroni L, Reis RL, Van Blitterswijk CA, Alves NM, Karperien M, Mano JF Chitosan (CHT)/poly(ɛ-caprolactone) (PCL) blend 3D fiber-mesh scaffolds were studied as possible support structures for articular cartilage tissue (ACT) repair. Micro-fibers were obtained by wet-spinning of three different polymeric solutions: 100:0 (100CHT), 75:25 (75CHT) and 50:50 (50CHT) wt.% CHT/PCL, using a common solvent solution of 100 vol.% of formic acid. Scanning electron microscopy (SEM) analysis showed a homogeneous surface distribution of PCL. PCL was well dispersed throughout the CHT phase as analyzed by differential scanning calorimetry and Fourier transform infrared spectroscopy. The fibers were folded into cylindrical moulds and underwent a thermal treatment to obtain the scaffolds. μCT analysis revealed an adequate porosity, pore size and interconnectivity for tissue engineering applications. The PCL component led to a higher fiber surface roughness, decreased the scaffolds swelling ratio and increased their compressive mechanical properties. Biological assays were performed after culturing bovine articular chondrocytes up to 21 days. SEM analysis, live-dead and metabolic activity assays showed that cells attached, proliferated, and were metabolically active over all scaffolds formulations. Cartilaginous extracellular matrix (ECM) formation was observed in all formulations. The 75CHT scaffolds supported the most neo-cartilage formation, as demonstrated by an increase in glycosaminoglycan production. In contrast to 100CHT scaffolds, ECM was homogenously deposited on the 75CHT and 50CHT scaffolds. Although mechanical properties of the 50CHT scaffold were better, the 75CHT scaffold facilitated better neo-cartilage formation. PMID: 20980050 [PubMed - as supplied by publisher] | | | | | | | | | | | | | | | | | | | | | | | | | Properties of hepatocyte-like cell clusters from human adipose tissue-derived mesenchymal stem cells. Tissue Eng Part C Methods. 2010 Aug;16(4):761-70 Authors: Okura H, Komoda H, Saga A, Kakuta-Yamamoto A, Hamada Y, Fumimoto Y, Lee CM, Ichinose A, Sawa Y, Matsuyama A There are only a few reports that describe the hepatocytic differentiation potential of human adipose tissue-derived mesenchymal stem cells (hADMSCs) and no reports that describe the in vivo functions of hepatocyte-like cells differentiated from somatic stem cells including hADMSCs. In this study, we established a new method for generation of functional hepatocyte-like cell clusters using floating culture method and induced functional hepatocyte-like cell clusters, which functioned effectively not only in vitro but also in vivo. The generated hepatocyte-like cell clusters were characterized by gene expression analysis, functional assays, and transplantation into non-obese diabetic severe combined immunodeficiency (NOD-SCID) mouse with chronic liver injury. The generated hepatocyte-like cell clusters expressed various genes normally found on mature hepatocytes. The cell clusters exhibited functional characteristics of hepatocytes: they expressed albumin, secreted urea, had cytochrome P450 activity, could take up low-density lipoprotein, and stored glycogen. Transplantation of these cell clusters into NOD-SCID mouse with chronic liver injury resulted in a significant improvement of serum albumin and total bilirubin levels. In summary, we established a new protocol for efficient induction of hADMSCs into functional hepatocyte-like cell clusters. PMID: 19839740 [PubMed - indexed for MEDLINE] | | | | | | | | | | | | | | | | | | | | | | | | | Bone formation on carbon nanotube composite. J Biomed Mater Res A. 2010 Oct 26; Authors: Bhattacharya M, Wutticharoenmongkol-Thitiwongsawet P, Hamamoto DT, Lee D, Cui T, Prasad HS, Ahmad M The effects of a layer-by-layer assembled carbon nanotube composite (CNT-comp) on osteoblasts in vitro and bone tissue in vivo in rats were studied. The effects of CNT-comp on osteoblasts were compared against the effects by commercially pure titanium (cpTi) and tissue culture dishes. Cell proliferation on the CNT-comp and cpTi were similar. However, cell differentiation, measured by alkaline phosphatase activity and matrix mineralization, was better on the CNT-comp. When implanted in critical-sized rat calvarial defect, the CNT-comp permitted bone formation and bone repair without signs of rejection or inflammation. These data indicate that CNT-comp may be a promising substrate for use as a bone implant or as a scaffold for tissue engineering. © 2010 Wiley Periodicals, Inc. J Biomed Mater Res Part A:, 2010. PMID: 20979130 [PubMed - as supplied by publisher] | | | | | | | | | | | | | | | | | | | | | | | | | Improved cellularization and angiogenesis using collagen scaffolds chemically conjugated with vascular endothelial growth factor. Acta Biomater. 2010 Oct 23; Authors: He Q, Zhao Y, Chen B, Xiao Z, Zhang J, Chen L, Chen W, Deng F, Dai J Much research has focused on developing vascular endothelial growth factor (VEGF) delivery systems for enhancing angiogenesis in wound repair and in tissue engineering. Collagen can be used as a delivery system because of its biocompatibility, but its fast degradation rate and limited affinity with growth factors are disadvantageous for maintaining enough growth factor concentration at injury sites. To enhance VEGF binding on collagen scaffolds and reduce the collagen degradation rate, we found a simple way to modify collagen porous scaffolds by chemical addition of sulfhydryl groups, which then allow both cross-linking of the collagen fibers with each other and the immobilization of more VEGF in the scaffold after treatment with Sulfo-SMCC. We demonstrated that cross-linking leaded to a slower degradation rate of the collagen scaffolds, while cellularization was improved both by cross-linking and by VEGF presence. On the other hand, angiogenesis was increased only moderately by cross-linking, but significantly more by the presence of immobilized VEGF. We conclude that collagen scaffolds chemically conjugated to VEGF by Traut's reagent and Sulfo-SMCC is an effective delivery system in wound repair and tissue engineering. PMID: 20977949 [PubMed - as supplied by publisher] | | | | | | | | | | | | | | | | | | | | | | | | | Three-dimensional laser micro- and nanostructuring of acrylated polyethylene glycol materials and evaluation of their cytoxicity for tissue engineering applications. Acta Biomater. 2010 Oct 23; Authors: Ovsianikov A, Malinauskas M, Schlie S, Chichkov B, Gittard S, Narayan R, Löbler M, Sternberg K, Schmitz KP, Haverich A The natural cell environment is characterised by complex three-dimensional structures, which contain features at multiple length scales. Many in vitro studies of cell behaviour in three dimensions rely on the availability of artificial scaffolds with controlled three-dimensional topologies. In this paper, we demonstrate fabrication of three-dimensional scaffolds for tissue engineering out of poly(ethylene glycol) diacrylate (PEGda) materials by means of two-photon polymerization (2PP). This laser nanostructuring approach offers unique possibilities for rapid manufacturing of three-dimensional structures with arbitrary geometries. The spatial resolution dependence on the applied irradiation parameters is investigated for two PEGda formulations, which are characterized by molecular weights of 302 and 742. We demonstrate that minimum feature sizes of 200 nm are obtained in both materials. In addition, an extensive study of the cytotoxicity of the material formulations with respect to photoinitiator type and photoinitiator concentration is undertaken. Aqueous extracts from photopolymerized PEGda samples indicate the presence of water-soluble molecules, which are toxic to fibroblasts. It is shown that sample aging in aqueous medium reduces the cytotoxicity of these extracts; this mechanism provides a route for biomedical applications of structures generated by 2PP microfabrication and photopolymerization technologies in general. Finally, a fully biocompatible combination of PEGda and a photoinitiator is identified. Fabrication of reproducible scaffold structures is very important for systematic investigation of cellular processes in three dimensions and for better understanding of in vitro tissue formation. The results of this work suggest that 2PP may be used to polymerize poly(ethylene glycol)-based materials into three-dimensional structures with well-defined geometries that mimic the physical and biological properties of native cell environments. PMID: 20977947 [PubMed - as supplied by publisher] | | | | | | | | | | | | | | | | | | | | | | | | | Short bouts of mechanical loading are as effective as dexamethasone at inducing matrix production by human bone marrow mesenchymal stem cell. Eur Cell Mater. 2010;20:45-57 Authors: Sittichokechaiwut A, Edwards JH, Scutt AM, Reilly GC Dexamethasone (Dex) is used widely to induce differentiation in human mesenchymal stem cells (hMSCs); however, using a pharmaceutical agent to stimulate hMSC differentiation is not the best choice for engineered tissue transplantation due to potential side-effects. The goal of the present study was to investigate the effects of dynamic compressive loading on differentiation and mineralized matrix production of hMSCs in 3D polyurethane scaffolds, using a loading regimen previously shown to stimulate mineralised matrix production of mature bone cells (MLO-A5). hMSCs were seeded in polyurethane scaffolds and cultured in standard culture media with or without Dex. Cell-seeded scaffolds were compressed at 5% global strain for 2 h on day 9 and then every 5 days in a media-filled sterile chamber. Samples were tested for mRNA expression of alkaline phosphatase (ALP), osteopontin (OPN), collagen type 1 (col 1) and runt-related transcription factor-2 (RUNX-212 h) after the first loading, cell viability by MTS assay and alkaline phosphatase activity at day 12 of culture and cell viability, collagen content by Sirius red and calcium content by alizarin red at day 24 of culture. Neither Dex nor loading had significant effects on cell viability. Collagen content was significantly higher (p<0.01) in the loaded group compared with the non-loaded group in all conditions. There was no difference in ALP activity or the amount of collagen and calcium produced between the non-loaded group supplemented with Dex and the loaded group without Dex. We conclude that dynamic loading has the ability to stimulate osteogenic differentiation of hMSC in the absence of glucocorticoids. PMID: 20648425 [PubMed - indexed for MEDLINE] | | | | | | | | | |  | |  | |  |
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