|  |  |  | | | | | pubmed: "regenerative medici... | | | | | | | | | | | | | | | | | | | Proliferation and mineralization of bone marrow cells cultured on macroporous hydroxyapatite scaffolds functionalized with collagen type I for bone tissue regeneration. J Biomed Mater Res A. 2010 Oct;95(1):1-8 Authors: Teixeira S, Fernandes MH, Ferraz MP, Monteiro FJ This study concerns the preparation and in vitro characterization of functionalized hydroxyapatite (HA) porous scaffolds, which are intended to be used as drug-delivery systems and bone-regeneration matrices. Hydroxyapatite scaffolds were prepared using the polymer replication method, and, after being submitted to a specific sintering cycle, collagen Type I was incorporated on the surface. After the coating procedure, collagen was crosslinked using the N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) conjugation method. In this study, hydroxyapatite scaffolds with uncrosslinked and crosslinked Type I collagen were evaluated. Cell morphology and deposition of extracellular matrix were assessed by scanning electron microscopy, whereas cell distribution was visualized by means of methylene blue staining. MTS and total DNA quantification assays were used to evaluate the viability and proliferation of human bone marrow cells cultured on all the materials for 28 days. Results showed that the cells were able to adhere, proliferate, and form a mineralized matrix on the surface of all the materials. Furthermore, the cells were able to spread from one pore to another and form cell clusters. The results show that these scaffolds are good candidates to serve as drug delivery vehicles and for tissue engineering purposes. (c) 2010 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2010. PMID: 20740596 [PubMed - in process] | | | | | | | | | | | | | | | | | | | | | | | | | Collagen oligomers modulate physical and biological properties of three-dimensional self-assembled matrices. Biopolymers. 2010 Aug 24; Authors: Bailey JL, Critser PJ, Whittington C, Kuske JL, Yoder MC, Voytik-Harbin SL Elucidation of mechanisms underlying collagen fibril assembly and matrix-induced guidance of cell fate will contribute to the design and expanded use of this biopolymer for research and clinical applications. Here, we define how type I collagen oligomers affect in-vitro polymerization kinetics as well as fibril microstructure and mechanical properties of formed matrices. Monomers and oligomers were fractionated from acid-solubilized pig skin collagen and used to generate formulations varying in monomer/oligomer content or average polymer molecular weight (AMW). Polymerization half-times decreased with increasing collagen AMW and closely paralleled lag times, indicating that oligomers effectively served as nucleation sites. Furthermore, increasing AMW yielded matrices with increased interfibril branching and had no correlative effect on fibril density or diameter. These microstructure changes increased the stiffness of matrices as evidenced by increases in both shear storage and compressive moduli. Finally, the biological relevance of modulating collagen AMW was evidenced by the ability of cultured endothelial colony forming cells to sense associated changes in matrix physical properties and alter vacuole and capillary-like network formation. This work documents the importance of oligomers as another physiologically-relevant design parameter for development and standardization of polymerizable collagen formulations to be used for cell culture, regenerative medicine, and engineered tissue applications. (c) 2010 Wiley Periodicals, Inc. Biopolymers, 2010. PMID: 20740490 [PubMed - as supplied by publisher] | | | | | | | | | | | | | | | | | | | | | | | | | Biological evaluation of solid freeformed, hard tissue scaffolds for orthopedic applications. J Appl Biomater Biomech. 2010 May-Aug;8(2):89-96 Authors: U Herath HM, Di Silvio L, Evans JR Purpose: Hydroxyapatite (HA) lattices were made by extrusion freeforming, a rapid prototyping process, and sintered to produce hard tissue scaffolds for bone regeneration. These highly reticulated lattice structures can be built directly from a computer design file which decides and controls their macroscopic shape, pore structure and size distribution. They are therefore defect-specific and show potential in tissue engineering for non-load bearing sites. Methods: Using a commercial human osteoblast-like cell line (HOS TE 85), biocompatibility was evaluated in an in vitro study. A high level of cell adhesion was evident by scanning electron microscopy on both convex and concave surfaces and the cell attachment was revealed at different depths into the scaffold. An AlamarBlue(R) assay was carried out to assess cell proliferation, which was further confirmed by quantifying total DNA concentration and total protein content. Results: The cell proliferation was significant and the pattern was comparable to that of the tissue culture control, ThermanoxTM. ALP activity and osteocalcin were quantified to evaluate the extent of cell differentiation, which confirmed the retention of the phenotype for the period studied. Mineralization of the matrix was determined via formation of nodules. Conclusions: HA scaffolds are non-toxic, able to maintain cell viability and support cell growth, proliferation, differentiation, and nodule formation. PMID: 20740471 [PubMed - in process] | | | | | | | | | | | | | | | | | | | | | | | | | Cyclic mechanical stimulation favors myosin heavy chain accumulation in engineered skeletal muscle constructs. J Appl Biomater Biomech. 2010 May-Aug;8(2):68-75 Authors: Candiani G, Riboldi SA, Sadr N, Lorenzoni S, Neuenschwander P, Montevecchi FM, Mantero S Purpose: Since stretching plays a key role in skeletal muscle tissue development in vivo, by making use of an innovative bioreactor and a biodegradable microfibrous scaffold (DegraPol(R)) previously developed by our group, we aimed to investigate the effect of mechanical conditioning on the development of skeletal muscle engineered constructs, obtained by seeding and culturing murine skeletal muscle cells on electrospun membranes. Methods: Following 5 days of static culture, skeletal muscle constructs were transferred into the bioreactor and further cultured for 13 days, while experiencing a stretching pattern adapted from the literature to resemble mouse development and growth. Sample withdrawal occurred at the onset of cyclic stretching and after 7 and 10 days. Myosin heavy chain (MHC) accumulation in stretched constructs (D) was evaluated by Western blot analysis and immunofluorescence staining, using statically cultured samples (S) as controls. Results: Western blot analysis of MHC on dynamically (D) and statically (S) cultured constructs at different time points showed that, at day 10, the applied stretching pattern led to an eight-fold increase in myosin accumulation in cyclically stretched constructs (D) with respect to the corresponding static controls (S). These results were confirmed by immunofluorescence staining of total sarcomeric MHC. Conclusions: Since previous attempts to reproduce skeletal myogenesis in vitro mainly suffered from the difficulty of driving myoblast development into an architecturally organized array of myosin expressing myotubes, the chance of inducing MHC accumulation via mechanical conditioning represents a significant step towards the generation of a functional muscle construct for skeletal muscle tissue engineering applications. PMID: 20740468 [PubMed - in process] | | | | | | | | | | | | | | | | | | | | | | | | | Polymer-based composite scaffolds for tissue engineering. J Appl Biomater Biomech. 2010 May-Aug;8(2):57-67 Authors: Gloria A, De Santis R, Ambrosio L Tissue engineering may be defined as the application of biological, chemical and engineering principles toward the repair, restoration or regeneration of living tissue using biomaterials, cells and biologically active molecules alone or in combinations. The rapid restoration of tissue biomechanical function represents a great challenge, highlighting the need to mimic tissue structure and mechanical behavior through scaffold designs. For this reason, several biodegradable and bioresorbable materials, as well as technologies and scaffold designs, have been widely investigated from an experimental and/or clinical point of view. Accordingly, this review aims at stressing the importance of polymer-based composite materials to make multifunctional scaffolds for tissue engineering, with a special focus on bone, ligaments, meniscus and cartilage. Moreover, polymer-based nanocomposites will also be briefly introduced as an interesting strategy to improve the biological and mechanical performances of polymer scaffolds, especially for bone tissue engineering. PMID: 20740467 [PubMed - in process] | | | | | | | | | | | | | | | | | | | | | | | | | Fabrication of chemically cross-linked porous gelatin matrices. J Appl Biomater Biomech. 2009 Sep-Dec;7(3):194-9 Authors: Bozzini S, Petrini P, Altomare L, Tanzi MC Purpose: The aim of this study was to chemically cross-link gelatin, by reacting its free amino groups with an aliphatic diisocyanate. Methods: To produce hydrogels with controllable properties, the number of reacting amino groups was carefully determined. Porosity was introduced into the gelatin-based hydrogels through the lyophilization process. Porous and non-porous matrices were characterized with respect to their chemical structure, morphology, water uptake and mechanical properties. Results: The physical, chemical and mechanical properties of the porous matrices are related to the extent of their cross-linking, showing that they can be controlled by varying the reaction parameters. Water uptake values (24 hours) vary between 160% and 200% as the degree of cross-linking increases. The flexibility of the samples also decreases by changing the extent of cross-linking. Young's modulus shows values between 0.188 KPa, for the highest degree, and 0.142 KPa for the lowest degree. Conclusions: The matrices are potential candidates for use as tissue-engineering scaffolds by modulating their physical chemical properties according to the specific application. PMID: 20740429 [PubMed - in process] | | | | | | | | | | | | | | | | | | | | | | | | | 3D fiber deposition technique to make multifunctional and tailor-made scaffolds for tissue engineering applications. J Appl Biomater Biomech. 2009 Sep-Dec;7(3):141-52 Authors: Gloria A, Russo T, De Santis R, Ambrosio L Tissue engineering represents an interesting approach which aims to create tissues and organs de novo. In designing scaffolds for tissue engineering applications, the principal goal is to mimic the function of the natural extracellular matrix, providing a temporary template for the growth of target tissues. For this reason, scaffolds should possess suitable mechanical properties and architecture to play their specific role. In this paper, limitations of conventional scaffold fabrication methods will be briefly introduced, and rapid prototyping techniques will be described as advanced processing methods to realize customized scaffolds with controlled internal microarchitecture. Among the rapid prototyping techniques, the potential and challenges of 3D fiber deposition to create multifunctional and tailor-made scaffolds will be reviewed. PMID: 20740423 [PubMed - in process] | | | | | | | | | | | | | | | | | | | | | | | | | High frequencies of leukemia stem cells in poor-outcome childhood precursor-B acute lymphoblastic leukemias. Leukemia. 2010 Aug 26; Authors: Morisot S, Wayne AS, Bohana-Kashtan O, Kaplan IM, Gocke CD, Hildreth R, Stetler-Stevenson M, Walker RL, Davis S, Meltzer PS, Wheelan SJ, Brown P, Jones RJ, Shultz LD, Civin CI In order to develop a xenograft model to determine the efficacy of new therapies against primary human precursor-B acute lymphoblastic leukemia (ALL) stem cells (LSCs), we used the highly immunodeficient non-obese diabetic (NOD).Cg-Prkdc(scid)IL2rg(tmlWjl)/SzJ (NOD-severe combined immune deficient (scid) IL2rg(-/-)) mouse strain. Intravenous transplantation of 2 of 2 ALL cell lines and 9 of 14 primary ALL cases generated leukemia-like proliferations in recipient mice by 1-7 months after transplant. Leukemias were retransplantable, and the immunophenotypes, gene rearrangements and expression profiles were identical or similar to those of the original primary samples. NOD-scid mice transplanted with the same primary samples developed similar leukemias with only a slightly longer latency than did NOD-scid-IL2Rg(-/-) mice. In this highly sensitive NOD-scid-IL2Rg(-/-)-based assay, 1-100 unsorted primary human ALL cells from five of five tested patients, four of whom eventually experienced leukemia relapse, generated leukemias in recipient mice. This very high frequency of LSCs suggests that a hierarchical LSC model is not valuable for poor-outcome ALL.Leukemia advance online publication, 26 August 2010; doi:10.1038/leu.2010.184. PMID: 20739953 [PubMed - as supplied by publisher] | | | | | | | | | | | | | | | | | | | | | | | | | From skin cells to hepatocytes: advances in application of iPS cell technology. J Clin Invest. 2010 Aug 25; Authors: Greenbaum LE The discovery several years ago that fibroblasts and other somatic cells from mice and humans can be reprogrammed to become inducible pluripotent stem (iPS) cells has created enthusiasm for their potential applications in regenerative medicine and for modeling human diseases. Two independent studies in this issue of the JCI provide evidence that iPS cells represent a promising source of hepatocytes for a wide range of applications, including cell transplantation, drug toxicity testing, patient-specific disease modeling, and even ex vivo gene therapy. But how far have we come? PMID: 20739747 [PubMed - as supplied by publisher] | | | | | | | | | | | | | | | | | | | | | | | | | Generation of Bioengineered Corneas with Decellularized Xenografts and Human Keratocytes. Invest Ophthalmol Vis Sci. 2010 Aug 25; Authors: Gonzalez-Andrades M, Cardona JD, Ionescu AM, Campos A, Perez MD, Alaminos M Purpose: Decellularization of animal corneas is a promising method for the development of artificial human corneas by tissue engineering. In this study, we have evaluated two different decellularization protocols to determine which one is able to best preserve the histological structure, composition and optical behavior of decellularized porcine corneas. Then, these corneas were recellularized with human keratocytes in order to obtain a partial human corneal substitute. Methods: We have applied two different decellularization protocols using NaCl and SDS to determine which one is able to best preserve the histological structure, composition and optical behavior of the decellularized corneas Then, those decellularized corneas that showed the most appropriate results were recellularized with human keratocytes and evaluated at the histological, biochemical and optical levels for use in regenerative medicine. Results: Our results showed that 1.5 M NaCl treatment of porcine corneas is able to generate an acellular corneal stroma with adequate histological and optical properties, and that human keratocytes are able to penetrate and spread within this scaffold with proper levels of cell differentiation. In contrast, 0.1% SDS treatment of porcine corneas resulted in high levels of fibrils disorganization and poor optical behavior of these corneas. Conclusions: In conclusion, we suggest that the decellularization of animal corneas using 1.5 M NaCl represents a useful method for the development of human bioengineered corneas with therapeutic potential. PMID: 20739475 [PubMed - as supplied by publisher] | | | | | | | | | | | | | | | | | | | | | | | | | Comparison of Candidate Materials for a Synthetic Osteo-Odonto Keratoprosthesis (OOKP) device. Invest Ophthalmol Vis Sci. 2010 Aug 25; Authors: Tan XW, Perera AP, Tan A, Tan DT, Khor KA, Beuerman RW, Mehta JS Purpose: Osteo-Odonto Keratoprosthesis (OOKP) is one of the most successful forms of keratoprosthesis surgery for end stage corneal and ocular surface disease. There is a lack of detailed comparison studies on the biocompatibilities of different materials used in keratoprosthesis. The aim of this investigation is to compare synthetic bioinert materials used for keratoprosthesis surgery with hydroxyapatite (HA) as a reference. Methods: Test materials were sintered titanium oxide (TiO2), aluminum oxide (Al2O3) and yttria-stabilized zirconia (YSZ) with density >95%. Bacteria adhesion on the substrates was evaluated using scanning electron microscopy and the spread plate METHOD: Surface properties of the implant discs were scanned using optical microscopy. Human keratocyte attachment and proliferation rates were assessed by cell counting and MTT assay at different time points. Morphological analysis and immunoblotting was used to evaluate focal adhesion formation while cell adhesion force was measured with a multimode atomic force microscope (AFM). Results: We found that bacterial adhesion on the TiO2, Al2O3, and YSZ surfaces were lower compared to that on HA substrates. TiO2 significantly promoted keratocytes proliferation and viability compared with HA, Al2O3, and YSZ. Immunofluorescent imaging analyses, immunoblotting and atomic force microscope measurement revealed that TiO2 surfaces enhanced cell spreading and cell adhesion compared to HA and Al2O3. Conclusions: TiO2 is the most suitable replacement candidate for use as skirt material as it enhanced cell functions and reduced bacterial adhesion. This would in turn enhance tissue integration and reduce device failure rates during keratoprosthesis surgery. PMID: 20739467 [PubMed - as supplied by publisher] | | | | | | | | | | | | | | | | | | | | | | | | | Biological characteristics of stem cells from foetal, cord blood and extraembryonic tissues. J R Soc Interface. 2010 Aug 25; Authors: Abdulrazzak H, Moschidou D, Jones G, Guillot PV Foetal stem cells (FSCs) can be isolated during gestation from many different tissues such as blood, liver and bone marrow as well as from a variety of extraembryonic tissues such as amniotic fluid and placenta. Strong evidence suggests that these cells differ on many biological aspects such as growth kinetics, morphology, immunophenotype, differentiation potential and engraftment capacity in vivo. Despite these differences, FSCs appear to be more primitive and have greater multi-potentiality than their adult counterparts. For example, foetal blood haemopoietic stem cells proliferate more rapidly than those found in cord blood or adult bone marrow. These features have led to FSCs being investigated for pre- and post-natal cell therapy and regenerative medicine applications. The cells have been used in pre-clinical studies to treat a wide range of diseases such as skeletal dysplasia, diaphragmatic hernia and respiratory failure, white matter damage, renal pathologies as well as cancers. Their intermediate state between adult and embryonic stem cells also makes them an ideal candidate for reprogramming to the pluripotent status. PMID: 20739312 [PubMed - as supplied by publisher] | | | | | | | | | | | | | | | | | | | | | | | | | Epigenetic control of Hox genes during neurogenesis, development, and disease. Ann Anat. 2010 Aug 6; Authors: Barber BA, Rastegar M The process of mammalian development is established through multiple complex molecular pathways acting in harmony at the genomic, proteomic, and epigenomic levels. The outcome is profoundly influenced by the role of epigenetics through transcriptional regulation of key developmental genes. Epigenetics refer to changes in gene expression that are inherited through mechanisms other than the underlying DNA sequence, which control cellular morphology and identity. It is currently well accepted that epigenetics play central roles in regulating mammalian development and cellular differentiation by dictating cell fate decisions via regulation of specific genes. Among these genes are the Hox family members, which are master regulators of embryonic development and stem cell differentiation and their mis-regulation leads to human disease and cancer. The Hox gene discovery led to the establishment of a fundamental role for basic genetics in development. Hox genes encode for highly conserved transcription factors from flies to humans that organize the anterior-posterior body axis during embryogenesis. Hox gene expression during development is tightly regulated in a spatiotemporal manner, partly by chromatin structure and epigenetic modifications. Here, we review the impact of different epigenetic mechanisms in development and stem cell differentiation with a clear focus on the regulation of Hox genes. PMID: 20739155 [PubMed - as supplied by publisher] | | | | | | | | | | | | | | | | | | | | | | | | | The mechanical characteristics and in vitro biocompatibility of poly(glycerol sebacate)-Bioglass((R)) elastomeric composites. Biomaterials. 2010 Aug 24; Authors: Liang SL, Cook WD, Thouas GA, Chen QZ Biodegradable elastomeric materials have gained much recent attention in the field of soft tissue engineering. Poly(glycerol sebacate) (PGS) is one of a new family of elastomers which are promising candidates used for soft tissue engineering. However, PGS has a limited range of mechanical properties and has drawbacks, such as cytotoxicity caused by the acidic degradation products of very soft PGS and degradation kinetics that are too fast in vivo to provide sufficient mechanical support to the tissue. However, the development of PGS/based elastomeric composites containing alkaline bioactive fillers could be a method for addressing these drawbacks and thus may pave the way towards wide clinical applications. In this study, we synthesized a new PGS composite system consisting of a micron-sized Bioglass((R)) filler. In addition to much improved cytocompatibility, the PGS/Bioglass((R)) composites demonstrated three remarkable mechanical properties. First, contrary to previous reports, the addition of microsized Bioglass((R)) increases the elongation at break from 160 to 550%, while enhancing the Young's modulus of the composites by up to a factor of four. Second, the modulus of the PGS/Bioglass((R)) composites drops abruptly in a physiological environment (culture medium), and the level of drop can be tuned such that the addition of Bioglass((R)) does not harden the composite in vivo and thus the desired compliance required for soft tissue engineering are maintained. Third, after the abrupt drop in modulus, the composites exhibited mechanical stability over an extended period. This latter observation is an important feature of the new composites, because they can provide reliable mechanical support to damaged tissues during the lag phase of the healing process. These mechanical properties, together with improved biocompatibility, make this family of composites better candidates than plastic and related composite biomaterials for the applications of tissue engineering. PMID: 20739061 [PubMed - as supplied by publisher] | | | | | | | | | | | | | | | | | | | | | | | | | Ultraviolet light crosslinking of poly(trimethylene carbonate) for elastomeric tissue engineering scaffolds. Biomaterials. 2010 Aug 23; Authors: Bat E, Kothman BH, Higuera GA, van Blitterswijk CA, Feijen J, Grijpma DW A practical method of photocrosslinking high molecular weight poly(trimethylene carbonate)(PTMC) is presented. Flexible, elastomeric and biodegradable networks could be readily prepared by UV irradiating PTMC films containing pentaerythritol triacrylate (PETA) and a photoinitiator. The network characteristics, mechanical properties, wettability, and in vitro enzymatic erosion of the photocrosslinked PTMC films were investigated. Densely crosslinked networks with gel contents up to 98% could be obtained in this manner. Upon photocrosslinking, flexible and tough networks with excellent elastomeric properties were obtained. To illustrate the ease with which the properties of the networks can be tailored, blends of PTMC with mPEG-PTMC or with PTMC-PCL-PTMC were also photocrosslinked. The wettability and the enzymatic erosion rate of the networks could be tuned by blending with block copolymers. Tissue engineering scaffolds were also fabricated using these flexible photocrosslinkable materials. After crosslinking, the fabricated PTMC-based scaffolds showed inter-connected pores and extensive microporosity. Human mesenchymal stem cell (hMSC) culturing studies showed that the photocrosslinked scaffolds prepared from PTMC and PTMC/PTMC-PCL-PTMC blends are well-suited for tissue engineering applications. PMID: 20739060 [PubMed - as supplied by publisher] | | | | | | | | | | | | | | | | | | | | | | | | | Redefining tissue engineering for nanomedicine in ophthalmology. Acta Ophthalmol. 2010 Aug 25; Authors: Ellis-Behnke R, Jonas JB Abstract. Working at the nanoscale means to completely rethink how to approach engineering in the body in general and in the eye in particular. In nanomedicine, tissue engineering is the ability to influence an environment either by adding, subtracting or manipulating that environment to allow it to be more conducive for its purpose. The goal is to function at the optimum state, or to return to that optimum state. Additive tissue engineering replaces cells or tissue, or tries to get something to grow that is no longer there. Arrestive tissue engineering tries to stop aberrant growth which, if left uncontrolled, would result in a decrease in function. Nano delivery of therapeutics can perform both additive and arrestive functions influencing the environment either way, depending on the targeting. By manipulating the environment at the nanoscale, the rate and distribution of healing can be controlled. It infers that potential applications of nanomedicine in ophthalmology include procedures, such as corneal endothelial cell transplantation, single retinal ganglion cell repair, check of retinal ganglion cell viability, building of nanofibre scaffolds, such as self-assembling peptides, to create a scaffold-like tissue-bridging structure to provide a framework for axonal regeneration in the case of optic nerve reconnection or eye transplantation, and ocular drug delivery. Examples of potential arrestive therapies include gene-related treatment modalities to inhibit intraocular neovascularization and to block retinal cell apoptosis. Looking towards the future, this review focuses on how nanoscale tissue engineering can be and is being used to influence that local environment. PMID: 20738260 [PubMed - as supplied by publisher] | | | | | | | | | | | | | | | | | | | | | | | | | [Assessment of gene therapy and regenerative therapy using radionuclide technique] Nippon Hoshasen Gijutsu Gakkai Zasshi. 2010 May 20;66(5):542-6 Authors: Inubushi M PMID: 20628223 [PubMed - indexed for MEDLINE] | | | | | | | | | | | | | | | | | | | | | | | | | Assessing the immunopotency of Toll-like receptor agonists in an in vitro tissue-engineered immunological model. Immunology. 2010 Jul;130(3):374-87 Authors: Ma Y, Poisson L, Sanchez-Schmitz G, Pawar S, Qu C, Randolph GJ, Warren WL, Mishkin EM, Higbee RG SUMMARY: The in vitro Peripheral Tissue Equivalent (PTE) module is a three-dimensional tissue-engineered endothelial cell/collagen matrix culture system, which has been reported to reproduce in vivo physiological conditions and which generates dendritic cells (DC) autonomously. In the present study, we used the PTE module to investigate the immunopotency of Toll-like receptor (TLR) agonists, including polyinosine-polycytidylic acid, Gardiquimod, CpG 2006 and lipopolysaccharide. Application of TLR agonists in the PTE module induced a wide range of cytokines, including interleukins 1alpha/beta, 6, 8 and 10 and tumour necrosis factor-alpha. Compared with traditional peripheral blood mononuclear cell (PBMC) cultures, the PTE module produced twofold to 100-fold higher levels of cytokine secretion, indicating that it can be a highly sensitive assay system. This increased sensitivity is the result of the natural synergy between the leucocytes and the endothelium. Furthermore, the application of TLR agonists, such as lipopolysaccharide and Gardiquimod, to the PTE module enhanced DC differentiation and promoted DC maturation, as indicated by up-regulated expression of CD83, CD86 and CCR7(CD197). In addition, functional assays indicated PTE-derived DC treated with Gardiquimod, a TLR-7 agonist, significantly augmented anti-tetanus toxoid antibody production. Interestingly, replacing PBMC with purified myeloid cells (CD33(+)) significantly reduced the responsiveness of the PTE module to TLR stimulation. The reduced sensitivity was partly the result of the removal of plasmacytoid DC that participated in the response to TLR stimulation and sensitization of the PTE module. Overall, the in vitro PTE module clearly demonstrated the effects of TLR agonists on DC generation, maturation and antigen-presenting capacity, and may serve as a sensitive and predictive test bed for the evaluation of adjuvant candidates. PMID: 20331478 [PubMed - indexed for MEDLINE] | | | | | | | | | | | | | | | | | | | | | | | | | Bladder wall transplantation--long-term survival of cells: implications for bioengineering and clinical application. Tissue Eng Part A. 2010 Jun;16(6):2121-7 Authors: Tanaka ST, Thangappan R, Eandi JA, Leung KN, Kurzrock EA Current bioengineered bladder wall substitutes include acellular scaffolds and grafts seeded with autologous cells. The transplanted cells on a seeded graft may regenerate and/or be replaced by cells of the patient's bladder. This may or may not be advantageous depending upon the underlying pathology. A theoretically perfect bioengineered graft would be intact bladder wall. To determine if such a graft is feasible and to study the cellular changes, we transplanted full-thickness bladder grafts from male inbred rats onto bladders of female syngeneic rats. Bladders were harvested at 1, 3, 6, 12, and 16 months after surgery and evaluated for histologic changes. Cell origin (male donor vs. female host) was determined with fluorescent in situ hybridization with unique probes for rat X and Y chromosomes. Urothelial hyperplasia, inflammation, and increased stromal thickness subsided down to control values by 6 months after surgery. At 16 months, graft muscle demonstrated persistence of male cells. On the other hand, graft urothelium was partially replaced by female host cells with a pattern suggestive of a hematogenous route rather than ingrowth from the host bladder. Bladder wall transplantation is feasible. The slow replacement of the transplanted urothelium and persistence of muscle may imply the same fate for engineered grafts. PMID: 20109058 [PubMed - indexed for MEDLINE] | | | | | | | | | | | | | | | | | | | | | | | | | Ex vivo construction of an artificial ocular surface by combination of corneal limbal epithelial cells and a compressed collagen scaffold containing keratocytes. Tissue Eng Part A. 2010 Jun;16(6):2091-100 Authors: Mi S, Chen B, Wright B, Connon CJ We have investigated the use of a laminin-coated compressed collagen gel containing corneal fibroblasts (keratocytes) as a novel scaffold to support the growth of corneal limbal epithelial stem cells. The growth of limbal epithelial cells was compared between compressed collagen gel and a clinically proven conventional substrate, denuded amniotic membrane (AM). Following compression of the collagen gel, encapsulated keratocytes remained viable and scanning electron microscopy showed that fibers within the compressed gel were dense, homogeneous, and similar in structure to those within denuded AM. Limbal epithelial cells were successfully expanded upon the compressed collagen, resulting in stratified layers of cells containing desmosome and hemidesmosome structures. The resulting corneal constructs of both the groups shared a high degree of transparency, cell morphology, and cell stratification. Similar protein expression profiles for cytokeratin 3 (CK3) and CK14 and no significant difference in CK12 mRNA expression levels by real-time polymerase chain reaction were also observed. This study provides the first line of evidence that a laminin-coated compressed collagen gel containing keratocytes can adequately support limbal epithelial cell expansion, stratification, and differentiation to a degree that is comparable to the leading conventional scaffold, denuded AM. PMID: 20109018 [PubMed - indexed for MEDLINE] | | | | | | | | | | | | | | | | | | | | | | | | | Recombinant gelatin microspheres: novel formulations for tissue repair? Tissue Eng Part A. 2010 Jun;16(6):1811-21 Authors: Tuin A, Kluijtmans SG, Bouwstra JB, Harmsen MC, Van Luyn MJ Microspheres (MSs) can function as multifunctional scaffolds in different approaches of tissue repair (TR), as a filler, a slow-release depot for growth factors, or a delivery vehicle for cells. Natural cell adhesion-supporting extracellular matrix components like gelatin are good materials for these purposes. Recombinant production of gelatin allows for on-demand design of gelatins, which is why we aim at developing recombinant gelatin (RG) MSs for TR. Two types of MSs (50 < Ø < 100 microm) were prepared by crosslinking two RGs, Syn-RG, and the arginine-glycine-aspartate-containing Hu-RG. The MSs were characterized, and their tissue reaction and degradation in rats was examined. Histological analysis of the explants after 14 and 28 days in vivo also showed that Syn-RG was degraded slower than Hu-RG, which correlated with the in vitro degradation assay. Hu-RG explants displayed more cellular ingrowth (60% vs. 15% for Syn-RG at day 14), which was associated with extracellular matrix deposition and vascularization. The infiltrating cells consisted of mainly macrophages, part of which fused to giant cells locally, and fibroblasts. No differences were found in matrix metalloproteinase mRNA levels, whereas gelatinase activity was clearly higher in Hu-RG explants. In conclusion, the in vitro and in vivo results of these novel formulations pave the way for cell- and/or factor-driven TR by these RG MSs. PMID: 20102269 [PubMed - indexed for MEDLINE] | | | | | | | | | | | | | | | | | | | | | | | | | Formation of hepatocyte spheroids with structural polarity and functional bile canaliculi using nanopillar sheets. Tissue Eng Part A. 2010 Jun;16(6):1983-95 Authors: Takahashi R, Sonoda H, Tabata Y, Hisada A We developed a method for controlling the spheroid formation of adult rat primary hepatocytes simply by optimizing the pillar diameters and patterns of nanopillar sheets. To investigate the effects of the pillar parameters on the spheroid formation, rat primary hepatocytes were cultured on nanopillar sheets with pillars that had one of five different diameters and that had been precoated with a solution containing one of two different concentrations of type I collagen. Spheroids with a compact morphology that were adhesive to the substratum and had an optimal size (50 to 100 microm) were obtained using a sheet with a pillar diameter of 2.0 microm that was precoated with 100 ng/mL of type I collagen solution. Immunohistochemistry revealed that the spheroids had a structure similar to that of native liver tissue. We then assessed the effect of overlaying reconstituted spheroids with Matrigel with the aim of achieving a simulated in vivo environment. The mRNA expression levels of MRP2, albumin, and P450-3A3 for spheroids determined by semiquantitative real-time PCR were significantly higher than those for spheroids cultured without the Matrigel overlay or for hepatocytes cultured using a conventional two-dimensional method. The spheroids obtained exhibited higher structural polarity and functional bile canaliculi compared with hepatocytes cultured using a conventional two-dimensional method. PMID: 20100035 [PubMed - indexed for MEDLINE] | | | | | | | | | | | | | | | | | | | | | | | | | Three-dimensional perfusion bioreactor culture supports differentiation of human fetal liver cells. Tissue Eng Part A. 2010 Jun;16(6):2007-16 Authors: Schmelzer E, Triolo F, Turner ME, Thompson RL, Zeilinger K, Reid LM, Gridelli B, Gerlach JC The ability of human fetal liver cells to survive, expand, and form functional tissue in vitro is of high interest for the development of bioartificial extracorporeal liver support systems, liver cell transplantation therapies, and pharmacologic models. Conventional static two-dimensional culture models seem to be inadequate tools. We focus on dynamic three-dimensional perfusion technologies and developed a scaled-down bioreactor, providing decentralized mass exchange with integral oxygenation. Human fetal liver cells were embedded in a hyaluronan hydrogel within the capillary system to mimic an in vivo matrix and perfusion environment. Metabolic performance was monitored daily, including glucose consumption, lactate dehydrogenase activity, and secretion of alpha-fetoprotein and albumin. At culture termination cells were analyzed for proliferation and liver-specific lineage-dependent cytochrome P450 (CYP3A4/3A7) gene expression. Occurrence of hepatic differentiation in bioreactor cultures was demonstrated by a strong increase in CYP3A4/3A7 gene expression ratio, lower alpha-fetoprotein, and higher albumin secretion than in conventional Petri dish controls. Cells in bioreactors formed three-dimensional structures. Viability of cells was higher in bioreactors than in control cultures. In conclusion, the culture model implementing three-dimensionality, constant perfusion, and integral oxygenation in combination with a hyaluronan hydrogel provides superior conditions for liver cell survival and differentiation compared to conventional culture. PMID: 20088704 [PubMed - indexed for MEDLINE] | | | | | | | | | | | | | | | | | | | | | | | | | Endothelial progenitor cells and mesenchymal stem cells seeded onto beta-TCP granules enhance early vascularization and bone healing in a critical-sized bone defect in rats. Tissue Eng Part A. 2010 Jun;16(6):1961-70 Authors: Seebach C, Henrich D, Kähling C, Wilhelm K, Tami AE, Alini M, Marzi I QUESTION/AIM: Lack of vessels indicates an insufficient nutritional supply of a bone graft and may limit the recruitment of bone-forming cells. Our aim was to evaluate the influence of endothelial progenitor cells (EPCs) alone or in combination with mesenchymal stem cells (MSCs) on early vascularization and bone healing in critical-sized defect (CSD) in vivo. METHODS: MSCs from human bone marrow and EPCs from buffy coat were used. A femoral CSD in adult athymic rats was created and stabilized by an external fixateur. The remaining defects were filled with fibronectin-coated beta-tricalcium phosphate (beta-TCP) granules, EPCs seeded on beta-TCP, MSCs seeded on beta-TCP, coculture of EPCs/MSCs seeded on beta-TCP, or autologous bone. Vascularization and bone formation were determined by immunohistology, microCT analysis, and biomechanical testing after 1, 4, and 8 weeks. RESULTS: Early vascularization was significantly improved in EPC/MSC group or EPC group, respectively. At 4 weeks bone formation increased significantly when the CSD was treated with coculture of MSCs/EPCs. Eight weeks after transplantation CSD showed significantly more bony bridgings and significantly increased ultimate load in the EPC/MSC group compared to the other groups. DISCUSSION: This cell approach suggests that there is a synergistic effect and that the initial stage of neovascularization by EPCs is considered to be crucial for complete bone regeneration in the late phase. PMID: 20088701 [PubMed - indexed for MEDLINE] | | | | | | | | | | | | | | | | | | | | | | | | | Scaffold-free in vitro arterial mimetics: the importance of smooth muscle-endothelium contact. Tissue Eng Part A. 2010 Jun;16(6):1901-12 Authors: Chaterji S, Park K, Panitch A We have developed an in vitro endothelial cell (EC)-smooth muscle cell (SMC) coculture platform that can mimic either the healthy or diseased state of blood vessels. Transforming growth factor-beta1 (TGF-beta1) and heparin were introduced to the SMC cultures to upregulate the SMC differentiation markers, alpha-smooth muscle actin (alpha-SMA) and calponin (homotypic model). Interestingly, seeding of near-confluent concentrations of ECs on the SMCs (heterotypic model) induced higher levels of alpha-SMA and calponin expression in the SMC cultures than did the addition of heparin and TGF-beta1 alone. The expression levels increased further on pretreating the SMCs with TGF-beta1 and heparin before adding a near-confluent monolayer of ECs. In contrast, seeding of sparse concentrations of ECs forced the SMCs into a more hyperplastic state as determined by alpha-SMA and calponin expression. This study highlights the importance of both soluble factors and EC seeding densities when considering culture conditions; in vivo SMCs are in close proximity with and interact with a monolayer of ECs. Our study suggests that this architecture is important for healthy vascular tissue function. In addition, it shows that disruption of this architecture can be used to mimic diseased states. As the EC-SMC coculture model can mimic either a diseased or a healthy blood vessel it may be useful as a test bed for evaluating cardiovascular therapeutics. PMID: 20088699 [PubMed - indexed for MEDLINE] | | | | | | | | | | | | | | | | | | | | | | | | | Impact of degradable macromer content in a poly(ethylene glycol) hydrogel on neural cell metabolic activity, redox state, proliferation, and differentiation. Tissue Eng Part A. 2010 Jun;16(6):1857-66 Authors: Lampe KJ, Bjugstad KB, Mahoney MJ Hydrogels that degrade at different rates were prepared by copolymerizing slowly degrading macromer poly(ethylene glycol) (PEG) dimethacrylate with a faster degrading macromer poly(lactic acid)-b-PEG-b-poly(lactic acid) dimethacrylate. A clinically relevant population of neural cells composed of differentiated neurons and multipotent precursor cells was cultured within hydrogels. Within 2 h after encapsulation, metabolic activity was higher in hydrogels prepared with increasing levels of degradable content. This improvement was accompanied by a reduction in intracellular redox state and an increase in the fraction of glutathione in the reduced state, both of which persisted throughout 7 days of culture and which may be the result of radical scavenging by lactic acid. Importantly, an increase in cellular proliferation was observed in gels prepared with increasing degradable macromer content after 7 days of growth without a shift in the cellular composition of the culture toward the glial cell phenotype. The findings of this study provide additional insight into the growth of neural cells in PEG-based hydrogels. Results suggest that lactic acid released during gel degradation may impact the function of encapsulated cells, a finding of general interest to biomaterials scientists who focus on the development of degradable polymers for cell culture and drug delivery devices. PMID: 20067398 [PubMed - indexed for MEDLINE] | | | | | | | | | | | | | | | | | | | | | | | | | Effect of dynamic stiffness of the substrates on neurite outgrowth by using a DNA-crosslinked hydrogel. Tissue Eng Part A. 2010 Jun;16(6):1873-89 Authors: Jiang FX, Yurke B, Schloss RS, Firestein BL, Langrana NA Central nervous system tissues, like other tissue types, undergo constant remodeling, which potentially leads to changes in their mechanical stiffness. Moreover, mechanical compliance of central nervous system tissues can also be modified under external load such as that experienced in traumatic brain or spinal cord injury, and during pathological processes. Thus, the neuronal responses to the dynamic stiffness of the microenvironment are of significance. In this study, we induced decrease in stiffness by using a DNA-crosslinked hydrogel, and subjected rat spinal cord neurons to such dynamic stiffness. The neurons respond to the dynamic cues as evidenced by the primary neurite structure, and the response from each neurite property (e.g., axonal length and primary dendrite number) is consistent with the behavior on static gels of same substrate rigidity, with one exception of mean primary dendrite length. The results on cell population distribution confirm the neuronal responses to the dynamic stiffness. Quantification on the focal adhesion kinase expression in the neuronal cell body on dynamic gels suggests that neurons also modify adhesion in coping with the dynamic stiffnesses. The results reported here extend the neuronal mechanosensing capability to dynamic stiffness of extracellular matrix, and give rise to a novel way of engineering neurite outgrowth in time dimension. PMID: 20067396 [PubMed - indexed for MEDLINE] | | | | | | | | | | | | | | | | | | | | | | | | | Porcine endothelial cells cocultured with smooth muscle cells became procoagulant in vitro. Tissue Eng Part A. 2010 Jun;16(6):1835-44 Authors: Pang Z, Niklason LE, Truskey GA Endothelial cell (EC) seeding represents a promising approach to provide a nonthrombogenic surface on vascular grafts. In this study, we used a porcine EC/smooth muscle cell (SMC) coculture model that was previously developed to examine the efficacy of EC seeding. Expression of tissue factor (TF), a primary initiator in the coagulation cascade, and TF activity were used as indicators of thrombogenicity. Using immunostaining, primary cultures of porcine EC showed a low level of TF expression, but a highly heterogeneous distribution pattern with 14% of ECs expressing TF. Quiescent primary cultures of porcine SMCs displayed a high level of TF expression and a uniform pattern of staining. When we used a two-stage amidolytic assay, TF activity of ECs cultured alone was very low, whereas that of SMCs was high. ECs cocultured with SMCs initially showed low TF activity, but TF activity of cocultures increased significantly 7-8 days after EC seeding. The increased TF activity was not due to the activation of nuclear factor kappa-B on ECs and SMCs, as immunostaining for p65 indicated that nuclear factor kappa-B was localized in the cytoplasm in an inactive form in both ECs and SMCs. Rather, increased TF activity appeared to be due to the elevated reactive oxygen species levels and contraction of the coculture, thereby compromising the integrity of EC monolayer and exposing TF on SMCs. The incubation of cocultures with N-acetyl-cysteine (2 mM), an antioxidant, inhibited contraction, suggesting involvement of reactive oxygen species in regulating the contraction. The results obtained from this study provide useful information for understanding thrombosis in tissue-engineered vascular grafts. PMID: 20055662 [PubMed - indexed for MEDLINE] | | | | | | | | | | | | | | | | | | | | | | | | | The effect of ex vivo dynamic loading on the osteogenic differentiation of genetically engineered mesenchymal stem cell model. J Tissue Eng Regen Med. 2010 Aug 26; Authors: Kimelman-Bleich N, Seliktar D, Kallai I, Helm GA, Gazit Z, Gazit D, Pelled G Mechanical loading has been described as a highly important stimulus for improvements in the quality and strength of bone. It has also been shown that mechanical stimuli can induce the differentiation of mesenchymal stem cells (MSCs) along the osteogenic lineage. We have previously demonstrated the potent osteogenic effect of MSCs engineered to overexpress the BMP2 gene. In this study we investigated the effect of mechanical loading on BMP2-expressing MSC-like cells, using a special bioreactor designed to apply dynamic forces on cell-seeded hydrogels. Cell viability, alkaline phosphatase (ALP) activity, BMP2 secretion and mineralized substance formation in the hydrogels were quantified. We found that cell metabolism increased as high as 6.8-fold, ALP activity by 12.5-fold, BMP2 secretion by 182-fold and mineralized tissue formation by 1.72-fold in hydrogels containing MSC-like cells expressing BMP2, which were cultured in the presence of mechanical loading. We have shown that ex vivo mechanical loading had an additive effect on BMP2-induced osteogenesis in genetically engineered MSC-like cells. These data could be valuable for bone tissue-engineering strategies of the future. Copyright (c) 2010 John Wiley & Sons, Ltd. PMID: 20740691 [PubMed - as supplied by publisher] | | | | | | | | | | | | | | | | | | | | | | | | | Polysaccharide-based materials for cartilage tissue engineering applications. J Tissue Eng Regen Med. 2010 Aug 26; Authors: Oliveira JT, Reis RL Tissue engineering was proposed approximately 15 years ago as an alternative and innovative way to address tissue regeneration problems. During the development of this field, researchers have proposed a variety of ways of looking into the regeneration and engineering of tissues, using different types of materials coupled with a wide range of cells and bioactive agents. This trilogy is commonly considered the basis of a tissue-engineering strategy, meaning by this the use of a support material, cells and bioactive agents. Different researchers have been adding to these basic approaches other parameters able to improve the functionality of the tissue-engineered construct, such as specific mechanical environments and conditioned gaseous atmospheres, among others. Nowadays, tissue-engineering principles have been applied, with different degrees of success, to almost every tissue lacking efficient regeneration ability and the knowledge and intellectual property produced since then has experienced an immense growth. Materials for regenerating tissues, namely cartilage, have also been continuously increasing and most of the theoretical requirements for a tissue engineering support have been addressed by a single material or a mixture of materials. Due to their intrinsic features, polysaccharides are interesting for cartilage tissue-engineering approaches and as a result their exploitation for this purpose has been increasing. The present paper intends to provide an overview of some of the most relevant polysaccharides used in cartilage tissue-engineering research. Copyright (c) 2010 John Wiley & Sons, Ltd. PMID: 20740689 [PubMed - as supplied by publisher] | | | | | | | | | | | | | | | | | | | | | | | | | Preparation of keratinocyte culture medium for the clinical applications of regenerative medicine. J Tissue Eng Regen Med. 2010 Aug 26; Authors: Takagi R, Yamato M, Murakami D, Kondo M, Yang J, Ohki T, Nishida K, Kohno C, Okano T Keratinocyte culture medium (KCM) has been used for the in vitro culture of keratinocytes and other types of epithelial cells, and the medium includes various ingredients. In this study, two modified KCMs were prepared. In the first, insulin, hydrocortisone and antibiotics that are normally included in KCM were replaced with clinically approved pharmaceutical agents, except transferrin and selenium; in the second, cholera toxin (CT) was replaced by L-isoproterenol (ISO). The modified KCMs were then compared to conventional KCM containing laboratory-grade reagents. Induced cell colony formations of canine oral mucosal epithelial cells cultured in both modified KCMs were found to be nearly equivalent to that in the control KCM, and there was no significant difference between the effect of CT and ISO. Canine oral mucosal cells proliferated to confluence in all three KCM formulations, with or without the use of 3T3 feeder layers. Cultured epithelial cells were harvested from temperature-responsive culture surfaces as an intact cell sheet, and the immunohistochemical analysis of the sheets showed that p63 and cytokeratin were expressed in the epithelial cell sheets cultured in all KCMs. Eventually, in the modified KCM formula, fetal bovine serum was replaced by autologous human serum, and the formula was found to be able to fabricate human oral mucosal epithelial cell sheets. These results indicated that the modified KCM was equally efficient as conventional KCM in the fabrication of transplantable stratified epithelial cell sheets. Copyright (c) 2010 John Wiley & Sons, Ltd. PMID: 20740688 [PubMed - as supplied by publisher] | | | | | | | | | | | | | | | | | | | | | | | | | Bridging the gap: Bone marrow aspiration concentrate reduces autologous bone grafting in osseous defects. J Orthop Res. 2010 Aug 25; Authors: Jäger M, Herten M, Fochtmann U, Fischer J, Hernigou P, Zilkens C, Hendrich C, Krauspe R Although autologous bone grafting represents an effective tool to induce osteogenic regeneration in local bone defects or pseudarthroses, it is associated with significant donor site morbidity and limited by the amount available for grafting. We investigate the potency of bone marrow aspiration concentrate (BMAC) to augment bone grafting and support bone healing. The functional and radiographic outcome of 39 patients with volumetric bone deficiencies treated with BMAC are presented and evaluated in a prospective clinical trial. A collagen sponge (Col) served as scaffold in 12 patients and a bovine hydroxyapatite (HA) was applied in the other 27 individuals. The minimal follow-up was 6 months. Clinical and radiographic findings were completed by in vitro data. All patients showed new bone formation in radiographs during follow-up. However, two patients underwent revision surgery due to a lack in bone healing. In contrast to the Col group, the postoperative bone formation appeared earlier in the HA group (HA group: 6.8 weeks vs. Col group 13.6 weeks). Complete bone healing was achieved in the HA group after 17.3 weeks compared to 22.4 weeks in the Col group. The average concentration factor of BMAC was 5.2 (SD 1.3). Flow cytometry confirmed the mesenchymal nature of the cells. Cells from BMAC created earlier and larger colonies of forming units fibroblasts (CFU-F) compared to cells from bone marrow aspirate. BMAC combined with HA can reduce the time needed for healing of bone defects when compared to BMAC in combination with collagen sponge. J. Orthop. Res. (c) 2010 Orthopaedic Research Society. PMID: 20740672 [PubMed - as supplied by publisher] | | | | | | | | | | | | | | | | | | | | | | | | | Protein polymer MRI contrast agents: Longitudinal analysis of biomaterials in vivo. Magn Reson Med. 2010 Aug 25; Authors: Karfeld-Sulzer LS, Waters EA, Kohlmeir EK, Kissler H, Zhang X, Kaufman DB, Barron AE, Meade TJ Despite recent advances in tissue engineering to regenerate biological function by combining cells with material supports, development is hindered by inadequate techniques for characterizing biomaterials in vivo. Magnetic resonance imaging is a tomographic technique with high temporal and spatial resolution and represents an excellent imaging modality for longitudinal noninvasive assessment of biomaterials in vivo. To distinguish biomaterials from surrounding tissues for magnetic resonance imaging, protein polymer contrast agents were developed and incorporated into hydrogels. In vitro and in vivo images of protein polymer hydrogels, with and without covalently incorporated protein polymer contrast agents, were acquired by magnetic resonance imaging. T(1) values of the labeled gels were consistently lower when protein polymer contrast agents were included. As a result, the protein polymer contrast agent hydrogels facilitated fate tracking, quantification of degradation, and detection of immune response in vivo. For the duration of the in vivo study, the protein polymer contrast agent-containing hydrogels could be distinguished from adjacent tissues and from the foreign body response surrounding the gels. The hydrogels containing protein polymer contrast agent have a contrast-to-noise ratio 2-fold greater than hydrogels without protein polymer contrast agent. In the absence of the protein polymer contrast agent, hydrogels cannot be distinguished by the end of the gel lifetime. Magn Reson Med, 2010. (c) 2010 Wiley-Liss, Inc. PMID: 20740653 [PubMed - as supplied by publisher] | | | | | | | | | | | | | | | | | | | | | | | | | Biocompatibility of poly(ethylene glycol)-based hydrogels in the brain: An analysis of the glial response across space and time. J Biomed Mater Res A. 2010 Oct;95(1):79-91 Authors: Bjugstad KB, Lampe K, Kern DS, Mahoney M Poly(ethylene glycol) or PEG-based hydrogels provide a useful methodology for tissue engineering and the controlled-release of drugs within the central nervous system (CNS). To be successful, the local neuroinflammatory response to an implant must be well understood. Toward this end, the focus was to examine the localized recruitment and activation of microglia and astrocytes following implantation of PEG-based hydrogels in the brain. Because they are of clinical relevance and may impact brain tissue differently, hydrogels with different mass loss profiles were examined. At all time points, a needle penetration in sham animals evoked a greater astrocytic response than hydrogel conditions. The astrocyte response that ensued varied with degradation rate. An attenuated response was present in more slowly degrading and nondegrading conditions. Relative to sham, hydrogel conditions attenuated the acute microglial response during the week after implant. By 56 days, microglial levels in shams decreased below the observed response in slowly degrading and nondegradable gels, which remained constant overtime. Although the inflammatory response to PEG-based hydrogels was complex depending on degradation rates, the magnitude of the acute microglia response and the long-term astrocyte response were attenuated suggesting the use of these materials for drug and cell delivery to the CNS. (c) 2010 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2010. PMID: 20740603 [PubMed - in process] | | | | | | | | | | | | | | | | | | | | | | | | | Electrospun nanofibrous matrix improves the regeneration of dense cortical bone. J Biomed Mater Res A. 2010 Oct;95(1):49-57 Authors: Cai YZ, Wang LL, Cai HX, Qi YY, Zou XH, Ouyang HW Numerous in vitro studies have indicated the potential of using electrospun nanofibrous scaffolds for tissue regeneration. However, few reports have demonstrated their utility in real tissue repair models. The present investigation tested the hypothesis that electrospun poly-L-lactic acid (PLLA) nanofibrous membrane leads to dense cortical bone regeneration and improves the efficacy of currently-used collagenous guided bone regeneration (GBR) membrane. In vitro, the function of bone marrow-derived mesenchymal stem cells (BMSCs) on nanofibrous scaffolds was evaluated. In an in vivo experiment, large bony defects were created in rabbit tibia and treated with a nanofiber-reinforced bilayer membrane, nanofibrous membrane, or collagenous membrane alone. Three and six weeks after operation, bone defect healing was assessed radiologically and histologically. In vitro differentiation studies showed that BMSCs had much higher expression of Runx2 and collagen type I, alpha 1 mRNAs, when cultured on nanofibrous scaffolds. The radiographic and histological data both showed that the group treated with bilayer membrane had more bony tissue formation at 3 weeks. Moreover, at 6 weeks, only the bilayer membrane-treated bone defects displayed better regeneration of cortical bone tissue, whereas in the other groups the defects were filled with spongy bone-like tissue. The results demonstrated that electrospun nanofibrous membrane improves the regeneration of cortical bone, suggesting that this type of membrane can be combined with current collagenous GBR membrane to improve guided bone regeneration technology. (c) 2010 Wiley Periodicals, Inc. J Biomed Mater Res Part A, 2010. PMID: 20740600 [PubMed - in process] | | | | | | | | | | | | | | | | | | | | | | | | | Early mammalian erythropoiesis requires the Dot1L methyltransferase. Blood. 2010 Aug 26; Authors: Feng Y, Yang Y, Ortega MM, Copeland JN, Zhang M, Jacob JB, Fields TA, Vivian JL, Fields PE Histone methylation is an important regulator of gene expression, and its coordinated activity is critical in complex developmental processes such as hematopoiesis. Disruptor of Telomere Silencing 1-Like (DOT1L) is a unique histone methyltransferase that specifically methylates histone H3 at lysine 79. We analyzed Dot1L mutant mice to determine the influence of this enzyme on embryonic hematopoiesis. The mutant mice developed more slowly than wildtype embryos and died between E10.5 and E13.5, displaying a striking anemia, especially apparent in the small vessels of the yolk sac. Further, a severe, selective defect in erythroid, but not myeloid differentiation was observed. The erythroid progenitors failed to develop normally, demonstrating retarded progression through the cell cycle, accumulation during the G(0)/G(1) stage, and a marked increase in apoptosis in response to erythroid growth factors. GATA2, a factor essential for early erythropoiesis, was significantly reduced in Dot1L-deficient cells, while expression of PU.1, a transcription factor that inhibits erythropoiesis and promotes myelopoiesis, was increased. These data suggest a model whereby DOT1L-dependent H3K79 methylation serves as a critical regulator of a differentiation switch during early hematopoiesis, regulating steady-state levels of GATA2 and PU.1 transcription and thus controlling the numbers of circulating erythroid and myeloid cells. PMID: 20798234 [PubMed - as supplied by publisher] | | | | | | | | | | | | | | | | | | | | | | | | | Human Artificial Chromosome with a Conditional Centromere for Gene Delivery and Gene Expression. DNA Res. 2010 Aug 26; Authors: Iida Y, Kim JH, Kazuki Y, Hoshiya H, Takiguchi M, Hayashi M, Erliandri I, Lee HS, Samoshkin A, Masumoto H, Earnshaw WC, Kouprina N, Larionov V, Oshimura M Human artificial chromosomes (HACs), which carry a fully functional centromere and are maintained as a single-copy episome, are not associated with random mutagenesis and offer greater control over expression of ectopic genes on the HAC. Recently, we generated a HAC with a conditional centromere, which includes the tetracycline operator (tet-O) sequence embedded in the alphoid DNA array. This conditional centromere can be inactivated, loss of the alphoid(tet)(-)(O) (tet-O HAC) by expression of tet-repressor fusion proteins. In this report, we describe adaptation of the tet-O HAC vector for gene delivery and gene expression in human cells. A loxP cassette was inserted into the tet-O HAC by homologous recombination in chicken DT40 cells following a microcell-mediated chromosome transfer (MMCT). The tet-O HAC with the loxP cassette was then transferred into Chinese hamster ovary cells, and EGFP transgene was efficiently and accurately incorporated into the tet-O HAC vector. The EGFP transgene was stably expressed in human cells after transfer via MMCT. Because the transgenes inserted on the tet-O HAC can be eliminated from cells by HAC loss due to centromere inactivation, this HAC vector system provides important novel features and has potential applications for gene expression studies and gene therapy. PMID: 20798231 [PubMed - as supplied by publisher] | | | | | | | | | | | | | | | | | | | | | | | | | Bioactive polymer/extracellular matrix scaffolds fabricated with a flow perfusion bioreactor for cartilage tissue engineering. Biomaterials. 2010 Aug 24; Authors: Liao J, Guo X, Grande-Allen KJ, Kasper FK, Mikos AG In this study, electrospun poly(varepsilon-caprolactone) (PCL) microfiber scaffolds, coated with cartilaginous extracellular matrix (ECM), were fabricated by first culturing chondrocytes under dynamic conditions in a flow perfusion bioreactor and then decellularizing the cellular constructs. The decellularization procedure yielded acellular PCL/ECM composite scaffolds containing glycosaminoglycan and collagen. PCL/ECM composite scaffolds were evaluated for their ability to support the chondrogenic differentiation of mesenchymal stem cells (MSCs) in vitro using serum-free medium with or without the addition of transforming growth factor-beta1 (TGF-beta1). PCL/ECM composite scaffolds supported chondrogenic differentiation induced by TGF-beta1 exposure, as evidenced in the up-regulation of aggrecan (11.6 +/- 3.8 fold) and collagen type II (668.4 +/- 317.7 fold) gene expression. The presence of cartilaginous matrix alone reduced collagen type I gene expression to levels observed with TGF-beta1 treatment. Cartilaginous matrix further enhanced the effects of growth factor treatment on MSC chondrogenesis as evidenced in the higher glycosaminoglycan synthetic activity for cells cultured on PCL/ECM composite scaffolds. Therefore, flow perfusion culture of chondrocytes on electrospun microfiber scaffolds is a promising method to fabricate polymer/extracellular matrix composite scaffolds that incorporate both natural and synthetic components to provide biological signals for cartilage tissue engineering applications. PMID: 20797784 [PubMed - as supplied by publisher] | | | | | | | | | | | | | | | | | | | | | | | | | Ultrasonography in Regenerative Injection (Prolotherapy) Using Dextrose, Platelet-rich Plasma, and Other Injectants. Phys Med Rehabil Clin N Am. 2010 Aug;21(3):585-605 Authors: Fullerton BD, Reeves KD Recent advances in ultrasound technology are leading physiatrists to new understandings of pain sources, new treatment options, and the ability to guide soft tissue interventions. This article examines the role of imaging ultrasound in diagnosing soft tissue injury and disease that may respond to regenerative medicine techniques (known as prolotherapy) using injectants such as dextrose, morrhuate sodium, or platelet-rich plasma. The current state of ultrasound evidence for these interventions is reviewed. Case examples assist in understanding clinical applications that currently outpace the evidence base. Development of quantitative ultrasound measures to objectively evaluate soft tissue organization is discussed. PMID: 20797551 [PubMed - as supplied by publisher] | | | | | | | | | | | | | | | | | | | | | | | | | Stem cells and the repair of radiation-induced salivary gland damage. Oral Dis. 2010 Aug 26; Authors: Coppes R, Stokman M Oral Diseases (2010) doi: 10.1111/j.1601-0825.2010.01723.x Hyposalivation underlying xerostomia after radiotherapy is still a major problem in the treatment of head and neck cancer. Stem cell therapy may provide a means to reduce radiation-induced hyposalivation and improve the quality of life of patients. This review discusses the current status in salivary gland stem cell research with respect to their potential to attenuate salivary gland dysfunction. Knowledge on the embryonic development, homeostasis and regeneration after atrophy of the salivary glands has provided important knowledge on the location of the salivary gland as well as on the factors that influence proliferation and differentiation. This knowledge has helped to locate, isolate and characterize cell populations that contain the salivary gland stem cell, although the exact tissue stem cell is still unidentified. The role that stem/progenitor cells play in the response to radiation and the factors that can influence stem/progenitor induced proliferation and differentiation are discussed. Finally, the mobilization and transplantation of stem cells and supportive cells and their potential to attenuate radiation-induced salivary gland damage are discussed. Based on the major advances made in the field of stem cell research, stem cell-based therapy has great potential to allow prevention or treatment of radiation-induced hyposalivation. PMID: 20796229 [PubMed - as supplied by publisher] | | | | | | | | | | | | | | | | | | | | | | | | | Penile Enhancement Using Autologous Tissue Engineering with Biodegradable Scaffold: A Clinical and Histomorphometric Study. J Sex Med. 2009 Oct 19; Authors: Perovic SV, Sansalone S, Djinovic R, Ferlosio A, Vespasiani G, Orlandi A Introduction. Autologous tissue engineering with biodegradable scaffolds is a new treatment option for real penile girth enhancement. Aim. The aim of this article is to evaluate tissue remodeling after penile girth enhancement using this technique. Methods. Between June 2005 and May 2007, a group of 12 patients underwent repeated penile widening using biodegradable scaffolds enriched with expanded autologous scrotal dartos cells. Clinical monitoring was parallel to histological investigation of tissue remodeling. During second surgical procedure, biopsies were obtained 10-14 months after first surgery (mean 12 months, N = 6) and compared with those obtained after 22-24 months (mean 23 months, N = 6), and control biopsies from patients who underwent circumcision (N = 5). Blind evaluation of histomorphometrical and immunohistochemical finding was performed in paraffin sections. Main Outcome Measurements. Penile girth gain in a flaccid state ranged between 1.5 and 3.8 cm (mean 2.1 +/- 0.28 cm) and in full erection between 1.2 and 4 cm (mean 1.9 +/- 0.28 cm). Patients' satisfaction, defined by a questionnaire, was good (25%) and very good (75%). Results. In biopsies obtained 10-14 months after first surgery, highly vascularized loose tissue with collagen deposition associated with small foci of mild chronic and granulomatous inflammation surrounding residual amorphous material was observed. Fibroblast-like hyperplasia and small vessel neoangiogenesis occurred intimately associated with the progressive growth of vascular-like structures from accumulation of CD34 and alpha-smooth muscle actin-positive cells surrounding residual scaffold-like amorphous material. Capillary neoangiogenesis occurred inside residual amorphous material. In biopsies obtained after 22-24 months, inflammation almost disappeared and tissue closely resembled that of the dartos fascia of control group. Conclusions. Autologous tissue engineering using expanded scrotal dartos cells with biodegradable scaffolds is a new and promising method for penile widening that generates progressive accumulation of stable collagen-rich, highly vascularized tissue matrix that closely resemble deep dartos fascia. Perovic SV, Sansalone S, Djinovic R, Ferlosio A, Vespasiani G, and Orlandi A. Penile enhancement using autologous tissue engineering with biodegradable scaffold: A clinical and histomorphometric study. J Sex Med **;**:**-**. PMID: 20796201 [PubMed - as supplied by publisher] | | | | | | | | | | | | | | | | | | | | | | | | | A Study on Repair of Porcine Articular Cartilage Defects With Tissue-Engineered Cartilage Constructed In Vivo By Composite Scaffold Materials. Ann Plast Surg. 2010 Aug 25; Authors: Lin PB, Ning LJ, Lian QZ, Xia Z, Xin Y, Sen BH, Fei NF The study was performed to find out a promising injectable composite scaffold for cartilage tissue engineering. By using a composite of allogenous cartilage microparticle acellular tissue matrix (CMACTM) and fibrin glue (Fg) as injectable scaffold materials, tissue-engineered cartilage was constructed in vivo, and the effects of which on the repair of porcine articular cartilage defects were observed.CMACTM was obtained from domestic pigs. The chondrocytes were prepared from experimental mini-type pigs and expanded in vitro. Fg was used as a scaffold material. The composite of CMACTM, second-passage chondrocytes, and Fg was replanted to the articular cartilage defective regions in autologous mini-type pig by injection. At 12 weeks after replantation, samples were collected and analyzed by general observation and histologic staining.The constructed tissue-engineered cartilage exhibited a good efficiency in the repair of articular cartilage defects. Cells in the constructed tissue-engineered cartilage grew well and were able to secrete cartilaginous matrix. The tissue-engineered cartilage showed a better biologic performance than the control.A composite of allogenous CMACTM and Fg was a promising injectable scaffold for cartilage tissue engineering, which could be used to repair articular cartilage defects by a minimally invasive procedure. PMID: 20798625 [PubMed - as supplied by publisher] | | | | | | | | | | | | | | | | | | | | | | | | | Translation of science to surgery: LINKING EMERGING CONCEPTS IN BIOLOGICAL CARTILAGE REPAIR TO SURGICAL INTERVENTION. J Bone Joint Surg Br. 2010 Sep;92(9):1195-202 Authors: Moran CJ, Shannon FJ, Barry FP, O'Byrne JM, O'Brien T, Curtin W Orthopaedic surgery is in an exciting transitional period as modern surgical interventions, implants and scientific developments are providing new therapeutic options. As advances in basic science and technology improve our understanding of the pathology and repair of musculoskeletal tissue, traditional operations may be replaced by newer, less invasive procedures which are more appropriately targeted at the underlying pathophysiology. However, evidence-based practice will remain a basic requirement of care. Orthopaedic surgeons can and should remain at the forefront of the development of novel therapeutic interventions and their application. Progression of the potential of bench research into an improved array of orthopaedic treatments in an effective yet safe manner will require the development of a subgroup of specialists with extended training in research to play an important role in bridging the gap between laboratory science and clinical practice. International regulations regarding the introduction of new biological treatments will place an additional burden on the mechanisms of this translational process, and orthopaedic surgeons who are trained in science, surgery and the regulatory environment will be essential. Training and supporting individuals with these skills requires special consideration and discussion by the orthopaedic community. In this paper we review some traditional approaches to the integration of orthopaedic science and surgery, the therapeutic potential of current regenerative biomedical science for cartilage repair and ways in which we may develop surgeons with the skills required to translate scientific discovery into effective and properly assessed orthopaedic treatments. PMID: 20798434 [PubMed - in process] | | | | | | | | | | | | | | | | | | | | | | | | | The Matrix-Binding Domain of Microfibril-Associated Glycoprotein-1 Targets Active Connective Tissue Growth Factor to a Fibroblast-Produced Extracellular Matrix. Macromol Biosci. 2010 Aug 26; Authors: Weinbaum JS, Tranquillo RT, Mecham RP It is advantageous to use biomaterials in tissue engineering that stimulate extracellular matrix (ECM) production by the cellular component. Connective tissue growth factor (CTGF) stimulates type I collagen (COL1A1) transcription, but is functionally limited as a free molecule. Using a matrix-binding domain (MBD) from microfibril-associated glycoprotein-1, the fusion protein MBD-CTGF was targeted to the ECM and tested for COL1A1 transcriptional activation. MBD-CTGF produced by the ECM-synthesizing fibroblasts, or provided exogenously, localized to the elastic fiber ECM. MBD-CTGF, but not CTGF alone, led to a two-fold enhancement of COL1A1 expression. This study introduces a targeting technology that can be used to elevate collagen transcription in engineered tissues and thereby improve tissue mechanics. PMID: 20799254 [PubMed - as supplied by publisher] | | | | | | | | | | | | | | | | | | | | | | | | | Melt Processing of Chitosan-Based Fibers and Fiber-Mesh Scaffolds for the Engineering of Connective Tissues. Macromol Biosci. 2010 Aug 26; Authors: Correlo VM, Costa-Pinto AR, Sol P, Covas JA, Bhattacharya M, Neves NM, Reis RL We report the production of chitosan-based fibers and chitosan fiber-mesh structures by melt processing (solvent-free) to be used as tissue-engineering scaffolds. The melt-based approach used to produce the scaffolds does not change their main characteristics, including the surface roughness and microporosity. The porosity, pore size, interconnectivity and mechanical performance of the scaffolds are all within the range required for various tissue-engineering applications. Biological assessments are performed in direct-contact assays. Cells are able to colonize the scaffold, including the inner porous structure. The cells show high indices of viability in all of the scaffold types. PMID: 20799253 [PubMed - as supplied by publisher] | | | | | | | | | | | | | | | | | | | | | | | | | A comparison of bioreactors for culture of fetal mesenchymal stem cells for bone tissue engineering. Biomaterials. 2010 Aug 24; Authors: Zhang ZY, Teoh SH, Teo EY, Khoon Chong MS, Shin CW, Tien FT, Choolani MA, Chan JK Bioreactors provide a dynamic culture system for efficient exchange of nutrients and mechanical stimulus necessary for the generation of effective tissue engineered bone grafts (TEBG). We have shown that biaxial rotating (BXR) bioreactor-matured human fetal mesenchymal stem cell (hfMSC) mediated-TEBG can heal a rat critical sized femoral defect. However, it is not known whether optimal bioreactors exist for bone TE (BTE) applications. We systematically compared this BXR bioreactor with three most commonly used systems: Spinner Flask (SF), Perfusion and Rotating Wall Vessel (RWV) bioreactors, for their application in BTE. The BXR bioreactor achieved higher levels of cellularity and confluence (1.4-2.5x, p < 0.05) in large 785 mm(3) macroporous scaffolds not achieved in the other bioreactors operating in optimal settings. BXR bioreactor-treated scaffolds experienced earlier and more robust osteogenic differentiation on von Kossa staining, ALP induction (1.2-1.6x, p < 0.01) and calcium deposition (1.3-2.3x, p < 0.01). We developed a Micro CT quantification method which demonstrated homogenous distribution of hfMSC in BXR bioreactor-treated grafts, but not with the other three. BXR bioreactor enabled superior cellular proliferation, spatial distribution and osteogenic induction of hfMSC over other commonly used bioreactors. In addition, we developed and validated a non-invasive quantitative micro CT-based technique for analyzing neo-tissue formation and its spatial distribution within scaffolds. PMID: 20739062 [PubMed - as supplied by publisher] | | | | | | | | | |  | |  | |  |
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