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| GMP-manufactured density gradient media for optimized mesenchymal stromal/stem cell isolation and expansion.
April 1, 2010 at 6:46 AM
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| | GMP-manufactured density gradient media for optimized mesenchymal stromal/stem cell isolation and expansion. Cytotherapy. 2010 Mar 30; Authors: Grisendi G, Annerén C, Cafarelli L, Sternieri R, Veronesi E, Luca Cervo G, Luminari S, Maur M, Frassoldati A, Palazzi G, Otsuru S, Bambi F, Paolucci P, Pierfranco C, Horwitz E, Dominici M Abstract Background aims. Bone marrow (BM) mesenchymal stromal/stem cells (MSC) are therapeutic tools in regenerative medicine and oncology. MSC isolation is often performed starting from a separation step based on research-grade 1.077 g/mL density gradient media (DGM). However, MSC clinical application should require the introduction of good manufacturing practice (GMP) reagents. We took advantage of two novel GMP DGM with densities of 1.077 and 1.073 g/mL (Ficoll-Paque PREMIUM and Ficoll-Paque PREMIUM 1.073, respectively) to test whether these reagents could isolate MSC efficiently while simultaneously comparing their performance. Methods. BM samples were processed using either 1.077 or 1.073 g/mL GMP DGM. BM mononucleated cell (MNC) fractions were analyzed for viability, immunophenotype, clonogenic potential, ex vivo expansion and differentiation potential. Results. No differences were noticed in cell recovery and viability between the groups. Fluorescence-acti! vated cell-sorting (FACS) analyzes on freshly isolated cells indicated that the 1.073 g/mL GMP DGM more efficiently depleted the CD45(+) fraction in comparison with 1.077 GMP DGM. Moreover, in the 1.073 group, fibroblastic colony-forming units (CFU-F) were 1.5 times higher and the final MSC yield 1.8 times increased after four passages. Both reagents isolated MSC with the expected phenotype; however, 1.073-isolated MSC showed a higher expression of CD90, CD146 and GD2. Additionally, MSC from both groups were capable of fully differentiating into bone, adipose cells and cartilage. Conclusions. Both GMP DGM enriched MSC from BM samples, suggesting that these reagents would be suitable for clinical-grade expansions. In addition, the density of 1.073 g/mL provides a significant advantage over 1.077 g/mL GMP DGM, impacting the quantity of MSC obtained and reducing the ex vivo expansion time for optimized cell-based clinical applications. PMID: 20353309 [PubMed - as supplied by publisher] | | |
| Engineered Disc-Like Angle-Ply Structures for Intervertebral Disc Replacement.
April 1, 2010 at 6:45 AM
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| | Engineered Disc-Like Angle-Ply Structures for Intervertebral Disc Replacement. Spine (Phila Pa 1976). 2010 Mar 27; Authors: Nerurkar NL, Sen S, Huang AH, Elliott DM, Mauck RL STUDY DESIGN.: To develop a construction algorithm in which electrospun nanofibrous scaffolds are coupled with a biocompatible hydrogel to engineer a mesenchymal stem cell (MSC)-based disc replacement. OBJECTIVE.: To engineer a disc-like angle-ply structure (DAPS) that replicates the multiscale architecture of the intervertebral disc. SUMMARY OF BACKGROUND DATA.: Successful engineering of a replacement for the intervertebral disc requires replication of its mechanical function and anatomic form. Despite many attempts to engineer a replacement for ailing and degenerated discs, no prior study has replicated the multiscale hierarchical architecture of the native disc, and very few have assessed the mechanical function of formed neo-tissues. METHODS.: A new algorithm for the construction of a disc analogue was developed, using agarose to form a central nucleus pulposus (NP) and oriented electrospun nanofibrous scaffolds to form the anulus fibrosus region (AF). Bovine ! MSCs were seeded into both regions and biochemical, histologic, and mechanical maturation were evaluated with in vitro culture. RESULTS.: We show that mechanical testing in compression and torsion, loading methods commonly used to assess disc mechanics, reveal equilibrium and time-dependent behaviors that are qualitatively similar to native tissue, although lesser in magnitude. Further, we demonstrate that cells seeded into both AF and NP regions adopt distinct morphologies that mirror those seen in native tissue, and that, in the AF region, this ordered community of cells deposit matrix that is organized in an angle-ply configuration. Finally, constructs demonstrate functional development with long-term in vitro culture. CONCLUSION.: These findings provide a new approach for disc tissue engineering that replicates multi-scale form and function of the intervertebral disc, providing a foundation from which to build a multi-scale, biologic, anatomically and hierarchically rel! evant composite disc analogue for eventual disc replacement. PMID: 20354467 [PubMed - as supplied by publisher] | | |
| Bioprinting Endothelial Cells With Alginate for 3D Tissue Constructs.
April 1, 2010 at 6:45 AM
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| | Bioprinting Endothelial Cells With Alginate for 3D Tissue Constructs. J Biomech Eng. 2009 Nov;131(11):111002 Authors: Khalil S, Sun W Advanced solid freeform fabrication (SFF) techniques have been an interest for constructing tissue engineered polymeric scaffolds because of its repeatability and capability of high accuracy in fabrication resolution at the scaffold macro- and microscales. Among many important scaffold applications, hydrogel scaffolds have been utilized in tissue engineering as a technique to confide the desired proliferation of seeded cells in vitro and in vivo into its architecturally porous three-dimensional structures. Such fabrication techniques not only enable the reconstruction of scaffolds with accurate anatomical architectures but also enable the ability to incorporate bioactive species such as growth factors, proteins, and living cells. This paper presents a bioprinting system designed for the freeform fabrication of porous alginate scaffolds with encapsulated endothelial cells. The bioprinting fabrication system includes a multinozzle deposition system that utilizes SFF! techniques and a computer-aided modeling system capable of creating heterogeneous tissue scaffolds. The manufacturing process is biologically compatible and is capable of functioning at room temperature and relatively low pressures to reduce the fluidic shear forces that could deteriorate biologically active species. The deposition system resolution is 10 mum in the three orthogonal directions XYZ and has minimum velocity of 100 mum/s. The ideal concentrations of sodium alginate and calcium chloride were investigated to determine a viable bioprinting process. The results indicated that the suitable fabrication parameters were 1.5% (w/v) sodium alginate and 0.5% (w/v) calcium chloride. Degradation studies via mechanical testing showed a decrease in the elastic modulus by 35% after 3 weeks. Cell viability studies were conducted on the cell encapsulated scaffolds for validating the bioprinting process and determining cell viability of 83%. This work exhibits the potential use! of accurate cell placement for engineering complex tissue reg! eneratio n using computer-aided design systems. PMID: 20353253 [PubMed - in process] | | |
| [Effect of angelica polysaccharides co-erythropoietin on JAK2/STAT5 signal transduction pathway in hematopoietic stem/progenitor cells]
April 1, 2010 at 6:45 AM
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| | [Effect of angelica polysaccharides co-erythropoietin on JAK2/STAT5 signal transduction pathway in hematopoietic stem/progenitor cells] Zhongguo Zhong Yao Za Zhi. 2009 Dec;34(24):3268-71 Authors: Hua Z, Song S, Luo C, Wang J, Wang Y OBJECTIVE: To investgate the signal transduction and regulation in erythropoiesis by angelica polysaccharides (APS) to clarify the mechanism for APS promoting hematopoiesis. METHOD: The mononuclear cells were isolated from foetus umbilic cord blood (mononuclear cells, MNCs), after MNCs were incubated in the presence of APS group (APS 200 mg x L(-1)) and control group for 24 h, the cells were stimulated with Epo (5 U x mL(-1)) for 0, 2, 5, 30 min, respectively. STAT5 was measured by ICC and laser confocal scanning microscope. JAK2, STAT5 in nucleus and cytoplasm were measured by western-blotting-ECL. RESULT: Angelica polysaccharide cooperated with Epo has significant impact on the expression of STAT5. The expression of STAT5 has significant difference between APS group and the control group at 4 time points. JAK2, STAT5 expressed in cytoplasm and nuclear of APS group significantly increased as compared to those of control group, and they expressed the strongest at ! 5 min. CONCLUSION: JAK2, STAT5 signal transduction molecule plays an important role in the effect of APS cooperated with Epo on promoting hematopoiesis. PMID: 20353016 [PubMed - in process] | | |
| Preparation and characterization of n-hydroxyapatite/PCL-pluronic-PCL nanocomposites for tissue engineering.
April 1, 2010 at 6:45 AM
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| | Preparation and characterization of n-hydroxyapatite/PCL-pluronic-PCL nanocomposites for tissue engineering. J Nanosci Nanotechnol. 2010 Feb;10(2):711-8 Authors: Fu S, Guo G, Wang X, Zhou L, Liu T, Dong P, Luo F, Gu Y, Shi X, Zhao X, Wei Y, Qian Z In this paper, a new kind of polymeric nanocomposite materials based on nano-hydroxyapatite (n-HA) and PCL-Pluronic-PCL (PCFC) copolymer were prepared by in situ combination method. Firstly, the PCFC copolymer was synthesized by ring-opening polymerization of epsilon-caprolactone initiated by Pluronic (PEG-PPG-PEG); Secondly, n-HA powder were combined with PCFC to form polymeric composites in the presence of hexamethylene diisocyanate (HDI). The obtained composites were characterized by 1H-NMR, FTIR, XRD, TEM, SEM, DTA/TGA, and tensile testing. The results revealed that n-HA could be dispersed into polymer matrix uniformly, and the n-HA/PCFC composite showed great mechanical properties when the content of n-HA was 10 wt%. The microstructure and thermal properties of the composites were discussed in the paper too. The experimental results suggested that this polymeric nanocomposite might have great potential application in the field of tissue engineering. PMID: 20352708 [PubMed - in process] | | |
| Tissue-engineered bilayered cell therapy for the treatment of oral mucosal defects: a case series.
April 1, 2010 at 6:45 AM
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| | Tissue-engineered bilayered cell therapy for the treatment of oral mucosal defects: a case series. Int J Periodontics Restorative Dent. 2010 Feb;30(1):31-9 Authors: Nevins ML In this case series, a tissue-engineered bilayered live cell therapy (LCT) is examined as an alternative to free gingival grafts in subjects with oral mucosal defects. Four patients with five sites requiring mucogingival surgery because of limited vestibular depth and limited keratinized oral mucosa were selected for treatment. All included patients had limited graft availability or preferred to avoid autogenous soft tissue grafting. The oral mucosal defects were treated with a tissue-engineered bilayered LCT, a circular disk of allogeneic live, human-derived, confluent, and bilayered keratinocyte and fibroblast cells. Sites were evaluated for wound healing, histologic appearance of the regenerated mucosa, and DNA persistence of the LCT. All sites healed uneventfully, with complete epithelialization by 14 days postoperative and no significant adverse events. The treated sites matched the surrounding tissues in both texture and color. Histologic samples revealed gi! ngival tissue architecture characterized by a parakeratinized epithelium with a very sparse and diffuse chronic inflammatory response within the connective tissue. DNA persistence indicated that the pairs of samples (swab and biopsy) collected from each subject displayed the same identifier DNA, with no evidence of any other source. Further research is needed to evaluate the utility of LCT in clinical practice. PMID: 20224829 [PubMed - indexed for MEDLINE] | | |
| Polysaccharide-modified synthetic polymeric biomaterials.
April 1, 2010 at 6:45 AM
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| | Polysaccharide-modified synthetic polymeric biomaterials. Biopolymers. 2010;94(1):128-40 Authors: Baldwin AD, Kiick KL This review presents an overview of polysaccharide-conjugated synthetic polymers and their use in tissue-engineered scaffolds and drug-delivery applications. This topic will be divided into four categories: (1) polymeric materials modified with non-mammalian polysaccharides such as alginate, chitin, and dextran; (2) polymers modified with mammalian polysaccharides such as hyaluronan, chondroitin sulfate, and heparin; (3) multi-polysaccharide-derivatized polymer conjugate systems; and (4) polymers containing polysaccharide-mimetic molecules. Each section will discuss relevant conjugation techniques, analysis, and the impact of these materials as micelles, particles, or hydrogels used in in-vitro and in-vivo biomaterial applications. PMID: 20091875 [PubMed - indexed for MEDLINE] | | |
| Elastin-like polypeptides: biomedical applications of tunable biopolymers.
April 1, 2010 at 6:45 AM
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| | Elastin-like polypeptides: biomedical applications of tunable biopolymers. Biopolymers. 2010;94(1):60-77 Authors: MacEwan SR, Chilkoti A Artificial repetitive polypeptides have grown in popularity as a bioinspired alternative to synthetic polymers. The genetically encoded synthesis, monodispersity, potential lack of toxicity, and biocompatibility are attractive features of these biopolymers for biological applications. Elastin-like polypeptides (ELPs) are one such class of biopolymers that are of particular interest because of their "smart"-stimuli responsive-properties. Herein, we discuss the genetically encoded design and recombinant synthesis of ELPs that enable precise control of their physicochemical properties and which have led to a wide range of biomedical applications of these biopolymers in the last decade. PMID: 20091871 [PubMed - indexed for MEDLINE] | | |
| Autologous Bone Marrow Derived Mononuclear Cell Therapy for Spinal Cord Injury: A Phase I/II Clinical Safety and Primary Efficacy Data.
April 1, 2010 at 6:27 AM
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| | Autologous Bone Marrow Derived Mononuclear Cell Therapy for Spinal Cord Injury: A Phase I/II Clinical Safety and Primary Efficacy Data. Exp Clin Transplant. 2009 Dec;7(4):241-8 Authors: Xiaofeng AA, Kumar SR, Narayanan R, Arul K, Baskaran M Objective: We sought to assess the safety and therapeutic efficacy of autologous human bone marrow derived mononuclear cell transplantation on spinal cord injury in a phase I/II, nonrandomized, open-label study, conducted on 297 patients. Materials and Methods: We transplanted unmanipulated bone marrow mononuclear cells through a lumbar puncture, and assessed the outcome using standard neurologic investigations and American Spinal Injury Association (ASIA) protocol, and with respect to safety, therapeutic time window, CD34-/+ cell count, and influence on sex and age. Results: No serious complications or adverse events were reported, except for minor reversible complaints. Sensory and motor improvements occurred in 32.6% of patients, and the time elapsed between the injury and the treatment considerably influenced the outcome of the therapy. The CD34-/+ cell count determined the state of improvement, or no improvement, but not the degree of improvement. No correlat! ion was found between level of injury and improvement, and age and sex had no role in the outcome of the cellular therapy. Conclusion: Transplant of autologous human bone marrow derived mononuclear cells through a lumbar puncture is safe, and one-third of spinal cord injury patients show perceptible improvements in the neurologic status. The time elapsed between injury and therapy and the number of CD34-/+ cells injected influenced the outcome of the therapy. PMID: 20353375 [PubMed - in process] | | |
| GMP-manufactured density gradient media for optimized mesenchymal stromal/stem cell isolation and expansion.
April 1, 2010 at 6:27 AM
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| | GMP-manufactured density gradient media for optimized mesenchymal stromal/stem cell isolation and expansion. Cytotherapy. 2010 Mar 30; Authors: Grisendi G, Annerén C, Cafarelli L, Sternieri R, Veronesi E, Luca Cervo G, Luminari S, Maur M, Frassoldati A, Palazzi G, Otsuru S, Bambi F, Paolucci P, Pierfranco C, Horwitz E, Dominici M Abstract Background aims. Bone marrow (BM) mesenchymal stromal/stem cells (MSC) are therapeutic tools in regenerative medicine and oncology. MSC isolation is often performed starting from a separation step based on research-grade 1.077 g/mL density gradient media (DGM). However, MSC clinical application should require the introduction of good manufacturing practice (GMP) reagents. We took advantage of two novel GMP DGM with densities of 1.077 and 1.073 g/mL (Ficoll-Paque PREMIUM and Ficoll-Paque PREMIUM 1.073, respectively) to test whether these reagents could isolate MSC efficiently while simultaneously comparing their performance. Methods. BM samples were processed using either 1.077 or 1.073 g/mL GMP DGM. BM mononucleated cell (MNC) fractions were analyzed for viability, immunophenotype, clonogenic potential, ex vivo expansion and differentiation potential. Results. No differences were noticed in cell recovery and viability between the groups. Fluorescence-acti! vated cell-sorting (FACS) analyzes on freshly isolated cells indicated that the 1.073 g/mL GMP DGM more efficiently depleted the CD45(+) fraction in comparison with 1.077 GMP DGM. Moreover, in the 1.073 group, fibroblastic colony-forming units (CFU-F) were 1.5 times higher and the final MSC yield 1.8 times increased after four passages. Both reagents isolated MSC with the expected phenotype; however, 1.073-isolated MSC showed a higher expression of CD90, CD146 and GD2. Additionally, MSC from both groups were capable of fully differentiating into bone, adipose cells and cartilage. Conclusions. Both GMP DGM enriched MSC from BM samples, suggesting that these reagents would be suitable for clinical-grade expansions. In addition, the density of 1.073 g/mL provides a significant advantage over 1.077 g/mL GMP DGM, impacting the quantity of MSC obtained and reducing the ex vivo expansion time for optimized cell-based clinical applications. PMID: 20353309 [PubMed - as supplied by publisher] | | |
| High recovery of mesenchymal progenitor cells with non-density gradient separation of human bone marrow.
April 1, 2010 at 6:27 AM
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| | High recovery of mesenchymal progenitor cells with non-density gradient separation of human bone marrow. Cytotherapy. 2010 Mar 30; Authors: Pozzo SD, Urbani S, Mazzanti B, Luciani P, Deledda C, Lombardini L, Benvenuti S, Peri A, Bosi A, Saccardi R Abstract Background aims. Bone marrow (BM) is the most used source of hemopoietic stem cells (HSC) and mesenchymal stromal cells (MSC) in both hematologic settings and regenerative medicine. We compared the feasibility and reproducibility of two gravity separation techniques, with or without the use of a density gradient, in terms of both hematopoietic and mesenchymal human BM progenitors. Methods. A total of 16 BM samples was processed to obtain mononuclear cells (MNC) and buffy coats (BC). The efficiency of the two procedures was evaluated by recovery of white blood cells (WBC), MNC and CD34(+) cells, clonogenic assays, red blood cell (RBC) depletion, cell viability, expression of embryonic transcriptional regulators and MSC assessment. Results. The two procedures yielded a comparable recovery of HSC. Non-density gradient separation (NDGS) of BM resulted in four times higher MSC recovery and higher expression of embryonic stem cell markers (Nanog and Sox2) compa! red with density-gradient separation (DGS). MSC derived from both procedures was comparable in terms of phenotype, differentiation and proliferation potential. Conclusions. NDGS is less time consuming, provides a better MSC enrichment and appears to be a suitable cell preparation method for clinical applications. PMID: 20353308 [PubMed - as supplied by publisher] | | |
| High-mobility group box 1 is involved in the initial events of early loss of transplanted islets in mice.
April 1, 2010 at 6:27 AM
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| | High-mobility group box 1 is involved in the initial events of early loss of transplanted islets in mice. J Clin Invest. 2010 Mar 1;120(3):735-43 Authors: Matsuoka N, Itoh T, Watarai H, Sekine-Kondo E, Nagata N, Okamoto K, Mera T, Yamamoto H, Yamada S, Maruyama I, Taniguchi M, Yasunami Y Islet transplantation for the treatment of type 1 diabetes mellitus is limited in its clinical application mainly due to early loss of the transplanted islets, resulting in low transplantation efficiency. NKT cell-dependent IFN-gamma production by Gr-1(+)CD11b(+) cells is essential for this loss, but the upstream events in the process remain undetermined. Here, we have demonstrated that high-mobility group box 1 (HMGB1) plays a crucial role in the initial events of early loss of transplanted islets in a mouse model of diabetes. Pancreatic islets contained abundant HMGB1, which was released into the circulation soon after islet transplantation into the liver. Treatment with an HMGB1-specific antibody prevented the early islet graft loss and inhibited IFN-gamma production by NKT cells and Gr-1(+)CD11b(+) cells. Moreover, mice lacking either of the known HMGB1 receptors TLR2 or receptor for advanced glycation end products (RAGE), but not the known HMGB1 receptor TLR4! , failed to exhibit early islet graft loss. Mechanistically, HMGB1 stimulated hepatic mononuclear cells (MNCs) in vivo and in vitro; in particular, it upregulated CD40 expression and enhanced IL-12 production by DCs, leading to NKT cell activation and subsequent NKT cell-dependent augmented IFN-gamma production by Gr-1(+)CD11b(+) cells. Thus, treatment with either IL-12- or CD40L-specific antibody prevented the early islet graft loss. These findings indicate that the HMGB1-mediated pathway eliciting early islet loss is a potential target for intervention to improve the efficiency of islet transplantation. PMID: 20124731 [PubMed - indexed for MEDLINE] | | | | 
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