Description

This authoritative peer-reviewed journal focuses on the engineering of new biologic tissues. The journal brings together scientific and medical experts in the fields of biomedical engineering, material science, molecular and cellular biology, and genetic engineering. The official journal of the Tissue Engineering Society.

Publisher details

Mary Ann Liebert

Publications in this journal

• Cardiomyocytes in vitro adhesion is actively influenced by biomimetic synthetic peptides for cardiac tissue engineering

  Authors: Gandaglia A, Huerta-Cantillo R, Comisso M, Danesin R, Ghezzo F, Naso F, Gastaldello A, Schittullo E, Buratto E, Spina M, Gerosa G, Dettin M

  Tissue Engineering.

• Best for Breast: Tissue Engineering Approaches for Breast Regeneration

  Authors: Amro A. Hussien, Meng Zhu, Noemi Kelemen, Vito Lotito, Alexander M. Seifalian

  Tissue Engineering.

• Dependence of neovascularization mechanisms on the molecular microenvironment.

  Authors: Eliane R Popa, Barry W A van der Strate, Linda A Brouwer, Henko Tadema, Martin Schipper, Brian Fernandes, Marc Hendriks, Marja J A van Luyn, Martin C Harmsen

  Tissue engineering. 13(12):2913-21.

  In vivo vascularization of implanted (bio)artificial constructs is essential for their proper function. Vascularization may rely on sprouting angiogenesis, vascular incorporation of boneIn vivo vascularization of implanted (bio)artificial constructs is essential for their proper function. Vascularization may rely on sprouting angiogenesis, vascular incorporation of bone marrow-derived endothelial cells (BMDECs), or both. Here we investigated the relative contribution of these 2 mechanisms to neovascularization in a mouse model of a foreign body reaction (FBR) to subcutaneously implanted Dacron and in hind limb ischemia (HLI) in relation to the molecular microenvironment at these neovascularization sites. Neovascularization was studied in C57Bl/6 mice reconstituted with enhanced green fluorescent protein (EGFP) transgenic bone marrow. Sprouting angiogenesis, detected using nuclear incorporation of bromodeoxyuridine in endothelial cells was present in both models, whereas vascular incorporation of EGFP(+) BMDECs was restricted to HLI. In HLI, the presence of a pro-angiogenic molecular microenvironment comprising vascular endothelial growth factor, fibroblast growth factor 2, and granulocyte colony-stimulating factor corroborated the importance of these factors for vascular BMDEC incorporation, whereas this microenvironment was absent in FBR. Enhanced mobilization of BMDECs by granulocyte-macrophage colony-stimulating factor administration or by combining HLI and FBR with Dacron did not induce incorporation of BMDECs in FBR neovessels. We conclude that the efficacy of BMDEC-based therapy is not generally warranted, but it depends on the molecular microenvironment in the targeted tissue.

• Design of silk-like biomaterials inspired by mussel-adhesive protein.

  Authors: Mingying Yang, Kazuo Yamauchi, Masato Kurokawa, Tetsuo Asakura

  Tissue engineering. 13(12):2941-7.

  To develop biomaterials for tissue engineering, a silk-like protein inspired by mussel-adhesive proteins (MAPs) was designed and prepared. The primary structure of this silk-like protein is designedTo develop biomaterials for tissue engineering, a silk-like protein inspired by mussel-adhesive proteins (MAPs) was designed and prepared. The primary structure of this silk-like protein is designed as TS[AKPSYPPTYKAS (GAGAGS)(3)](10) by combining the sequences (GAGAGS)(3), the crystalline region of Bombyx mori silk fibroin, and AKPSYPPTYK, the adhesive sequence of MAP from Mytilus edulis. This protein was synthesized by the genetic engineering method. Solid-state (13)C NMR spectra showed that this silk-like protein adopts flexible conformation due to introduction of the sequence AKPSYPPTYK. Cell assay indicated that this silk-like protein has significantly higher cell adhesion activities in response to normal human dermal fibroblasts (NHDFs) than Pronectin F, which is available as commercialized cell-adhesive silk-like protein. Thus, combination of remarkably high cell-adhesive activity from MAP with superiority of silk fibroin provides potentiality for application to the field of biomaterials.

• Microvascular transplantation after acute myocardial infarction.

  Authors: Benjamin R Shepherd, James B Hoying, Stuart K Williams

  Tissue engineering. 13(12):2871-9.

  The primary objective of this study was to evaluate epicardial transplantation of an intact microvascular network for treatment of myocardial ischemia in a murine model of acute myocardialThe primary objective of this study was to evaluate epicardial transplantation of an intact microvascular network for treatment of myocardial ischemia in a murine model of acute myocardial infarction. We describe transplantation of an intact microvascular network constructed from isolated microvascular segments stabilized in a 3-dimensional matrix to the epicardial surface after acute myocardial infarction. This microvascular graft was implanted as a patch on the epicardium of mice after left coronary artery ligation. After 14 and 28 days of implantation, left ventricular (LV) function was assessed and grafts evaluated via histology and cytochemistry. Inosculation of microvessels within the graft with host coronary microcirculation occurred as early as 7 days after initial tissue grafting. Morphologic evaluation of the grafts revealed arterioles, venules, capillaries, and erythrocytes within vascular lumina. Control grafts of collagen alone remained avascular. LV infarct size was smaller, and LV function improved in treated animals. Engraftment of whole microvascular units can be achieved to support cell-assisted vascular remodeling. Microvascular grafts may provide therapeutic benefit as a primary treatment or serve as a microvascular platform for cardiac repair and regeneration.

• A novel time-varying poly lactic-co glycolic acid external sheath for vein grafts designed under physiological loading.

  Authors: Sophia W Liao, Xiao Lu, Andrew J Putnam, Ghassan S Kassab

  Tissue engineering. 13(12):2855-62.

  Changes in dimensional and mechanical properties of degradable sheaths in poly lactic-co glycolic acid (PLGA) have been researched extensively. Composite PLGA having variable resorption rates inChanges in dimensional and mechanical properties of degradable sheaths in poly lactic-co glycolic acid (PLGA) have been researched extensively. Composite PLGA having variable resorption rates in multiple layers under physiological loading has not been reported. Our novel design of a PLGA sheath is composed of 3 layers with different degradation rates (i.e., the innermost layer degrades the fastest, followed by the middle, while the outer layer degrades the slowest). In the presence of physiological luminal pressure, diameter is greater, thickness is less, resorption rate is greater, pore size is greater, and incremental modulus is greater than in nonpressurized sheaths. Furthermore, the ratio of the pore size to the sheath radius affects the dimensional changes of the sheath in the radial direction. In addition to changing the pore size-to-sheath radius ratio, the dimensional changes can be manipulated by choosing different glycolic and lactic acid ratios for the different layers. The application of this novel PLGA design for gradual arterialization of vein grafts is contemplated.

• Bioreactor for application of subatmospheric pressure to three-dimensional cell culture.

  Authors: Robert P Wilkes, Amy K McNulty, Teri D Feeley, Marisa A Schmidt, Kris Kieswetter

  Tissue engineering. 13(12):3003-10.

  Vacuum-assisted closure (VAC) negative pressure wound therapy (NPWT) is a highly successful and widely used treatment modality for wound healing, although no apparatus exists to monitor the effectsVacuum-assisted closure (VAC) negative pressure wound therapy (NPWT) is a highly successful and widely used treatment modality for wound healing, although no apparatus exists to monitor the effects of subatmospheric pressure application in vitro. Such an apparatus is desirable to better understand the biological effects of this therapy and potentially improve upon them. This article describes the development and validation of a novel bioreactor that permits such study. Tissue analogues consisting of 3-dimensional fibroblast-containing fibrin clots were cultured in off-the-shelf disposable cell culture inserts and multi-well plates that were integrated into the bioreactor module. Negative pressure dressings, commercialized for wound therapy, were placed on top of the culture, and subatmospheric pressure was applied to the dressing. Cultures were perfused with media at controlled physiologic wound exudate flow rates. The design of this bioreactor permits observation of the culture using an inverted microscope in brightfield and fluorescence modes and sustained incubation of the system in a 5% carbon dioxide atmosphere. This closed-system mimics the wound micro-environment under VAC NPWT. Matrix compression occurs as the subatmospheric pressure draws the dressing material down. At the contact zone, surface undulations were clearly evident on the fibroblast-containing tissue analogues at 24 h and appeared to correspond to the dressing microstructure. The bioreactor design, consisting of sterilizable machined plastics and disposable labware, can be easily scaled to multiple units. Validation experiments show that cell survival in this system is comparable with that seen in cells grown in static tissue culture. After application of VAC NPWT, cell morphology changed, with cells appearing thicker and with an organized actin cytoskeleton. The development and validation of this new culture system establishes a stable platform for in vitro investigations of subatmospheric pressure application to tissues.

• In vitro expansion of adipose-derived adult stromal cells in hypoxia enhances early chondrogenesis.

  Authors: Yue Xu, Preeti Malladi, Michael Chiou, Elena Bekerman, Amato J Giaccia, Michael T Longaker

  Tissue engineering. 13(12):2981-93.

  Cartilage is an avascular tissue, and chondrocytes in vivo experience a severely hypoxic environment. Using a defined in vitro model of early chondrogenesis, we attempted to enrich for cells with anCartilage is an avascular tissue, and chondrocytes in vivo experience a severely hypoxic environment. Using a defined in vitro model of early chondrogenesis, we attempted to enrich for cells with an enhanced ability for chondrogenic differentiation by pre-exposure of mouse adipose-derived adult stromal cells (ADASs) to a hypoxic (2% oxygen) environment. ADASs were subsequently expanded in 2% or 21% oxygen environments, resulting in 2 groups of cells, and then early chondrogenic differentiation was induced at 21% oxygen tension using a 3-dimensional micromass culture system. ADAS chondrogenesis was assessed using Alcian Blue staining for proteoglycans and quantification of sulfated glycosaminoglycans. Osteogenesis of the 2 cell groups was also studied. Two percent oxygen tension profoundly increased the proliferation of ADASs. ADASs expanded in 2% oxygen tension exhibited enhanced early chondrogenic differentiation and diminished osteogenesis, suggesting that the reduced oxygen environment may favor chondroprogenitors. Gene expression analysis suggested that matrix metalloproteinase synthesis was inhibited in cells expanded in 2% oxygen. Furthermore, re-oxygenation of the 2% oxygen-expanded ADASs before differentiation did not significantly affect early chondrogenesis. Thus, priming ADASs with 2% oxygen may have selected for chondrogenic progenitors with an enhanced ability to survive and differentiate. This study is relevant for the future application of cell-based therapies involving cartilage tissue regeneration.

• Cartilage reshaping via in vitro mechanical loading.

  Authors: Gregory M Williams, Jessica W Lin, Robert L Sah

  Tissue engineering. 13(12):2903-11.

  Shaped cartilage grafts can be used in the restoration of injured joints and the reconstruction of deformities of the head and neck. This study describes a novel method for altering cartilage shape,Shaped cartilage grafts can be used in the restoration of injured joints and the reconstruction of deformities of the head and neck. This study describes a novel method for altering cartilage shape, based on the hypothesis that mechanical loading coupled with in vitro tissue growth and remodeling facilitates tissue reshaping. Static bending deformations were imposed on strips of immature articular cartilage, and retention of the imposed shape and structural and biochemical measures of growth were assessed after 2, 4, and 6 days of incubation. The results show that mechanical reshaping of tissue is feasible, because shape retention was greater than 86% after 6 days of culture. The imposed mechanical deformations had little effect on measures of tissue viability or growth within the 6-day culture period. The addition of cycloheximide to the culture medium only slightly reduced the ability to reshape these tissues, but cycloheximide plus a lower culture temperature of 4 degrees C markedly inhibited the reshaping response. These results suggest a limited role for chondrocyte biosynthesis but a potentially important role for metabolic reactions in the cartilage matrix in the reshaping process. The ability to modulate cartilage shape in vitro may prove useful for tissue engineering of shaped cartilage grafts.

• Development of custom-built bone scaffolds using mesenchymal stem cells and apatite-wollastonite glass-ceramics.

  Authors: Jennifer A Dyson, Paul G Genever, Kenneth W Dalgarno, David J Wood

  Tissue engineering. 13(12):2891-901.

  There is a clinical need for new bone replacement materials that combine long implant life with complete integration and appropriate mechanical properties. We have used human mesenchymal stem cellsThere is a clinical need for new bone replacement materials that combine long implant life with complete integration and appropriate mechanical properties. We have used human mesenchymal stem cells (MSCs) to populate porous apatite-wollastonite (A-W) glass-ceramic scaffolds produced by the layer manufacturing technique, selective laser sintering, to create custom-built bone replacements. Confocal and scanning electron microscopy were used to determine optimal seeding densities and to demonstrate that MSCs adhered and retained viability on the surface of A-W scaffolds over a culture period of 21 days. We found a significant increase in the number of MSCs growing on the scaffolds over 7 days. Using bromodeoxyuridine incorporation we demonstrated that MSCs proliferated on the scaffolds. Using real-time PCR we analyzed the expression of the osteogenic markers alkaline phosphatase, collagen type-I, Cbfa-1, osteocalcin, osteonectin, and osteopontin by MSCs cultured in the absence of osteogenic supplements. The expression of the osteogenic markers by MSCs was equivalent to or significantly greater on A-W scaffolds than on tissue culture plastic. We also identified significantly higher alkaline phosphatase activity on A-W compared to a commercial calcium phosphate scaffold. These results indicate for the first time the biocompatibility and osteo-supportive capacity of A-W scaffolds and their potential as patient-specific bone replacement materials.

• Suspension of bone marrow-derived undifferentiated mesenchymal stromal cells for repair of superficial digital flexor tendon in race horses.

  Authors: Simone Pacini, Silvia SPINABELLA, Luisa Trombi, Rita Fazzi, Sara Galimberti, Francesca Dini, Fabio Carlucci, Mario Petrini

  Tissue engineering. 13(12):2949-55.

  It has been proven that mesenchymal stromal cells (MSCs) can differentiate into tenocytes. Attempts to repair tendon lesions have been performed, mainly using scaffold carriers in experimentalIt has been proven that mesenchymal stromal cells (MSCs) can differentiate into tenocytes. Attempts to repair tendon lesions have been performed, mainly using scaffold carriers in experimental settings. In this article, we describe the clinical use of undifferentiated MSCs in racehorses. Significant clinical recovery was achieved in 9 of 11 horses evaluated using ultrasound analysis and their ability to return to racing. Our results show that the suspension of a small number of undifferentiated MSCs may be sufficient to repair damaged tendons without the use of scaffold support. Ultrasound scanning showed that fibers were correctly oriented. By using undifferentiated cells, no ectopic bone deposition occurred. A sufficient number of cells was recovered for therapeutic purposes in all but 1 case. We suggest that the use of autologous MSCs is a safe therapeutic method for treating incompletely (i.e., not full-thickness) damaged tendons.

• Strategic directions in tissue engineering.

  Authors: Peter C Johnson, Antonios G Mikos, John P Fisher, John A Jansen

  Tissue engineering. 13(12):2827-37.

  The field of tissue engineering is developing rapidly. Given its ultimate importance to clinical care, the time is appropriate to assess the field's strategic directions to optimize research andThe field of tissue engineering is developing rapidly. Given its ultimate importance to clinical care, the time is appropriate to assess the field's strategic directions to optimize research and development activities. To characterize strategic directions in tissue engineering, a distant but reachable clinical goal was proposed and a worldwide body of 24 leaders in tissue engineering was queried systematically to determine the best paths toward that goal. Using a modified Hoshin process, we identified 14 critical activity categories and then stratified them by their immediate priority for the field. The result of the analysis illustrates a highly interdependent set of activities that are dominated by the need for an understanding of angiogenesis, stem cell science, and the utilization of molecular biology and systems biology tools to enable a deeper comprehension of tissue development and control.

• Regulable Vascular Endothelial Growth Factor(165) Overexpression by Ex Vivo Expanded Keratinocyte Cultures Promotes Matrix Formation, Angiogenesis, and Healing in Porcine Full-Thickness Wounds.

  Authors: Stijn Dickens, Pieter Vermeulen, Benoit Hendrickx, Stefaan Van den Berge, Jan J Vranckx

  Tissue engineering.

  The intricate wound repair process involves the interplay of numerous cells and proteins. Using a porcine full-thickness wound (FTW) healing model, we hypothesized that the ex vivo gene transfer ofThe intricate wound repair process involves the interplay of numerous cells and proteins. Using a porcine full-thickness wound (FTW) healing model, we hypothesized that the ex vivo gene transfer of vascular endothelial growth factor (VEGF)-transfected basal keratinocyte (KC) cell suspensions may generate cross-talk and induce matrix formation, angiogenesis, and accelerated healing. Moreover, to regulate overexpression of isoform 165 of VEGF and its effect on healing, we introduced a tetracycline (TC)-inducible gene switch in the expression plasmid. Autologous basal KCs were cultivated from the porcine donor and transfected using cationic liposomes. A dose-response curve was established to determine optimal activation of the gene switch by TC. In vivo, FTWs were treated with VEGF-transfected KCs and controls. Wound fluids were collected daily and examined using enzyme-linked immunosorbent assay. Biopsies were evaluated using hematoxylin and eosin and immunostaining for fibronectin, CD144, and lectin BS-1. In vitro, highest regulable VEGF(165)-expression was obtained with 1 mug/mL of TCs. In vivo, after induction of the gene switch by adding 1 mug/mL of TCs to the FTW, we obtained upregulated VEGF(165) levels and enhanced fibronectin deposition and found more endothelial cell tubular formations and higher rates of reepithelialization than in controls. This ex vivo gene transfer model may serve as a platform for vascular induction in full-thickness tissue repair.

• Integrated 3-dimensional expansion and osteogenic differentiation of murine embryonic stem cells.

  Authors: Wesley L Randle, Jae Min Cha, Yu-Shik Hwang, K L Andrew Chan, Sergei G Kazarian, Julia M Polak, Athanasios Mantalaris

  Tissue engineering. 13(12):2957-70.

  Embryonic stem cell (ESC) culture is fragmented and laborious and involves operator decisions. Most protocols consist of 3 individual steps: maintenance, embryoid body (EB) formation, andEmbryonic stem cell (ESC) culture is fragmented and laborious and involves operator decisions. Most protocols consist of 3 individual steps: maintenance, embryoid body (EB) formation, and differentiation. Integration will assist automation, ultimately aiding scale-up to clinically relevant numbers. These problems were addressed by encapsulating undifferentiated murine ESCs (mESCs) in 1.1% (w/v) low-viscosity alginic acid, 0.1% (v/v) porcine gelatin hydrogel beads (d = 2.3 mm). Six hundred beads containing 10,000 mESCs per bead were cultured in a 50-mL high-aspect-ratio vessel bioreactor. Bioreactor cultures were rotated at 17.5 revolutions per min, cultured in maintenance medium containing leukemia inhibitory factor for 3 days, replaced with EB formation medium for 5 days followed by osteogenic medium containing L-ascorbate-2-phosphate (50 microg/mL), beta-glycerophosphate (10 mM), and dexamethasone (1 microM) for an additional 21 days. After 29 days, 84 times as many cells per bead were observed and mineralized matrix was formed within the alginate beads. Osteogenesis was confirmed using von Kossa, Alizarin Red S staining, alkaline phosphatase activity, immunocytochemistry for osteocalcin, OB-cadherin, collagen type I, reverse transcriptase polymerase chain reaction, microcomputed tomography (micro-computed tomography) and Fourier transform infrared spectroscopic imaging. This simplified, integrated, and potentially scaleable methodology could enable the production of 3-demensional mineralized tissue from ESCs for potential clinical applications.

• Applications of MEMS technologies in tissue engineering.

  Authors: Christopher M Puleo, Hsin-Chih Yeh, Tza-Huei Wang

  Tissue engineering. 13(12):2839-54.

  The success of therapeutic strategies within the fields of regenerative medicine, including tissue engineering, biomaterials engineering, and cell and tissue transplantation science, relies onThe success of therapeutic strategies within the fields of regenerative medicine, including tissue engineering, biomaterials engineering, and cell and tissue transplantation science, relies on researchers' understanding of the complex cellular microenvironments that occur within functional tissue. Microfabricated biomedical platforms provide tools for researchers to study cellular response to various stimuli with micro- and nanoscale spatial control. Initial studies utilizing relatively passive means of microenvironmental control have provided the fundamental knowledge required to begin to design microculture platforms that closely mimic these biological systems. In this review, we discuss second-generation cell and tissue culture platforms that utilize active components, borrowed from work in the development of microelectromechanical systems (MEMS). These microsystems offer the unprecedented opportunity to fabricate culture platforms designed to match tissue-specific growth parameters. In addition, the adoption of MEMS components opens up the door for future integration with the burgeoning field of microanalytical systems, providing analytical platforms that retain the sensitivity and resolution required within low-volume, microfluidic culture technologies.

• In vitro assessment of axonal growth using dorsal root ganglia explants in a novel three-dimensional collagen matrix.

  Authors: Ahmet Bozkurt, Gary A Brook, Sven Moellers, Franz Lassner, Bernd Sellhaus, Joachim Weis, Michael Woeltje, Julian Tank, Christina Beckmann, Paul Fuchs, Leon Olde Damink, Frank Schügner, Ingo Heschel, Norbert Pallua

  Tissue engineering. 13(12):2971-9.

  The goal of this study was the development of a bioartificial nerve guide to induce axonal regeneration in the peripheral nervous system (PNS). In this in vitro study, the ability of a novel,The goal of this study was the development of a bioartificial nerve guide to induce axonal regeneration in the peripheral nervous system (PNS). In this in vitro study, the ability of a novel, 3-dimensional (3D), highly oriented, cross-linked porcine collagen scaffold to promote directed axonal growth has been studied. Collagen nerve guides with longitudinal guidance channels were manufactured using a series of chemical and mechanical treatments with a patented unidirectional freezing process, followed by freeze-drying (pore sizes 20-50 microm). Hemisected rat dorsal root ganglia (DRG) were positioned such that neural and non-neural elements could migrate into the collagen scaffold. After 21 days, S100-positive Schwann cells (SCs) migrated into the scaffold and aligned within the guidance channels in a columnar fashion, resembling "Bands of Büngner." Neurofilament-positive axons (mean length +/- SD 756 microm +/- 318 microm, maximum 1496 microm) from DRG neurons entered the scaffold where the growth within the guidance channels was closely associated with the oriented SCs. This study confirmed the importance of SCs in the regeneration process (neurotrophic theory). The alignment of SCs within the guidance channels supported directional axonal growth (contact guidance theory). The microstructural properties of the scaffold (open, porous, longitudinal pore channels) and the in vitro data after DRG loading (axonal regeneration along migrated and columnar-aligned SCs resembling "Band of Büngner") suggest that this novel oriented 3D collagen scaffold serves as a basis for future experimental regeneration studies in the PNS.

• Fabrication of an optimal urethral graft using collagen-sponge tubes reinforced with Copoly(L-lactide/epsilon-caprolactone) fabric.

  Authors: Isao Kanatani, Akihiro Kanematsu, Yasuyuki Inatsugu, Masaaki Imamura, Hiromitsu Negoro, Noriyuki Ito, Shingo Yamamoto, Yasuhiko Tabata, Yoshito Ikada, Osamu Ogawa

  Tissue engineering. 13(12):2933-40.

  An ideal biomaterial for urethral reconstruction has not been developed. To create a urethral graft biomaterial with optimal biodegradability and biocompatibility, aAn ideal biomaterial for urethral reconstruction has not been developed. To create a urethral graft biomaterial with optimal biodegradability and biocompatibility, a copoly(L-lactide/epsilon-caprolactone) [P(LA/CL)] fabric tube was combined with a type I collagen sponge. The P(LA/CL) fibers were knitted into a vascular stent style (Type 1) or weaved into a mesh style (Type 2) to prepare P(LA/CL) tubes. The tubes were dipped in aqueous collagen solution and lyophilyzed to prepare the P(LA/CL)-collagen sponge graft. The grafts were applied to a 1.5-cm rabbit urethral defect (n = 14 for each condition), and tissue repair was evaluated using urethrographical, urethroscopical, and histological examination 1, 3, and 6 months after surgery. Although epithelialization was observed after 1 month in all Type 1 grafts, stenoses, fistulae, or stone formation was seen in 7 of the rabbits. In some cases, P(LA/CL) fibers prolapsed into the urethral lumen, causing stone formation. Only 3 rabbits survived for 6 months, and 2 of these had stenoses. For the Type 2 graft, all urethras were patent, without fistulae or stenoses, over the entire observation period. Histologically, urethral structure was disorganized for the Type 1 graft, whereas the urethral tissue on the Type 2 graft was slightly fibrotic but completely epithelialized and supported by a regenerated smooth muscle layer at 6 months. These findings suggest that creation of a scaffold suitable for urethral tissue regeneration will depend not only on the biomaterial composition, but also on the fabrication technique.

• Membranes from acrylonitrile-based polymers for selective cultivation of human keratinocytes.

  Authors: Gregor Boese, Christiane Trimpert, Wolfgang Albrecht, Günter Malsch, Thomas Groth, Andreas Lendlein

  Tissue engineering. 13(12):2995-3002.

  A cell carrier made from synthetic material supporting selective growth of keratinocytes is a promising approach to avoid the phenomenon of fibroblast overgrowth during in vitro culture of skinA cell carrier made from synthetic material supporting selective growth of keratinocytes is a promising approach to avoid the phenomenon of fibroblast overgrowth during in vitro culture of skin substitutes. Therefore, we investigated polymer membranes made of polyacrylonitrile and copolymers of acrylonitrile and N-vinylpyrrolidone (NVP) for their ability to support selectively the growth of keratinocytes. It was found that a copolymer with an NVP-content of 30% (NVP30) supports growth of human keratinocyte cell line (HaCaT) cells and inhibits fibroblast growth under serum-containing conditions. Cell proliferation of HaCaT cells was measured over 14 days. If both cell types were cultured under serum-free conditions for initial adhesion over 6 h on these NVP30 polymers, they adhered to the same extent. Long-term experiments over 7 days were performed as a coculture of both cell types showing that HaCaT cells had a growth advantage that seems to be related to the paracrine activity of contaminating fibroblasts. As a result, confluent layers of HaCaT cells were obtained with small numbers of remaining fibroblasts. The new poly [acrylonitrile-co(NVP) membranes seem to be a promising culture system for the production of epidermal transplants.

• Neonatal pig liver-derived progenitors for insulin-producing cells: an in vitro study.

  Authors: Leda Racanicchi, Giuseppe Basta, Pia Montanucci, Lucia Guido, Alessandra Pensato, Valentina Conti, Riccardo Calafiore

  Tissue engineering. 13(12):2923-31.

  Beta (beta)-cell replacement represents an attractive approach for the possible cure of type 1 insulin-dependent diabetes mellitus (IDDM). In a search for potential sources of insulin-secreting cellsBeta (beta)-cell replacement represents an attractive approach for the possible cure of type 1 insulin-dependent diabetes mellitus (IDDM). In a search for potential sources of insulin-secreting cells for IDDM substitution therapy, we have focused on the neonatal pig liver, which is putatively enriched in multipotent stem cells. We then isolated cells measuring 10 to 15 microm in diameter, identified as small cells, characterized by a high proliferation rate and positive staining for immature liver and pancreatic endocrine cell markers (i.e., insulin and pancreatic duodenal homeobox). The ability of these cells to transdifferentiate into pancreatic beta-like cells under culture conditions with exendin-4 (Ex-4) or high glucose concentration was examined. We observed that insulin secretion was not physiological in basal conditions, although it became responsive to glucose after 5 days of exposure to Ex-4. This beta-cell-like phenotype remained physiologically stable, even after stimulus withdrawal. Based on these observations, we contend that the proposed cell and tissue model might offer several advantages as a candidate for substitution cell therapy in IDDM, because the neonatal pig liver seems enriched in cells, with a mixed pancreas-liver phenotype, that are easier to purify and grow in culture and are more functional than other beta-like cells upon in vitro single short-term stimulation challenge.

• Editorial: advancing tissue engineering and regenerative medicine.

  Authors: Kiki B Hellman, Robert M Nerem

  Tissue engineering. 13(12):2823-4.

• Vascularized adipose tissue grafts from human mesenchymal stem cells with bioactive cues and microchannel conduits.

  Authors: Michael S Stosich, Barb Bastian, Nicholas W Marion, Paul A Clark, Gwendolen Reilly, Jeremy J Mao

  Tissue engineering. 13(12):2881-90.

  Vascularization is critical to the survival of engineered tissues. This study combined biophysical and bioactive approaches to induce neovascularization in vivo. Further, we tested the effects ofVascularization is critical to the survival of engineered tissues. This study combined biophysical and bioactive approaches to induce neovascularization in vivo. Further, we tested the effects of engineered vascularization on adipose tissue grafts. Hydrogel cylinders were fabricated from poly(ethylene glycol) diacrylate (PEG) in four configurations: PEG alone, PEG with basic fibroblast growth factor (bFGF), microchanneled PEG, or both bFGF-adsorbed and microchanneled PEG. In vivo implantation revealed no neovascularization in PEG, but substantial angiogenesis in bFGF-adsorbed and/or microchanneled PEG. The infiltrating host tissue consisted of erythrocyte-filled blood vessels lined by endothelial cells, and immunolocalized to vascular endothelial growth factor (VEGF). Human mesenchymal stem cells were differentiated into adipogenic cells, and encapsulated in PEG with both microchanneled and adsorbed bFGF. Upon in vivo implantation subcutaneously in immunodeficient mice, oil red O positive adipose tissue was present and interspersed with interstitial fibrous (IF) capsules. VEGF was immunolocalized in the IF capsules surrounding the engineered adipose tissue. These findings suggest that bioactive cues and/or microchannels promote the genesis of vascularized tissue phenotypes such as the tested adipose tissue grafts. Especially, engineered microchannels may provide a generic approach for modifying existing biomaterials by providing conduits for vascularization and/or diffusion.

• Tissue engineering of skeletal muscle.

  Authors: Wentao Yan, Sheela George, Upinder Fotadar, Natalia Tyhovych, Angela Kamer, Michael J Yost, Robert L Price, Charles R Haggart, Jeffrey W Holmes, Louis Terracio

  Tissue engineering. 13(11):2781-90.

  Loss of skeletal muscle profoundly affects the health and well-being of patients, and there currently is no way to replace lost muscle. We believe that a key step in the development of a prosthesisLoss of skeletal muscle profoundly affects the health and well-being of patients, and there currently is no way to replace lost muscle. We believe that a key step in the development of a prosthesis for reconstruction of dysfunctional muscular tissue is the ability to reconstitute the in vivo-like 3-dimensional (3D) organization of skeletal muscle in vitro with isolated satellite cells. In our present proof of principle studies, we have successfully constructed a multilayered culture of skeletal muscle cells, derived from neonatal satellite cells, that are distributed in a 3D pattern of organization that mimics many of the features of intact tissue. These multilayered cultures are composed of elongated multinucleated myotubes that are MyoD positive. Histological studies indicate that the multiple layers of myotubes can be distinguished. Expression of muscle-specific markers such as myosin heavy chain, dystrophin, integrin alpha-7, alpha-enolase, and beta-enolase was detected using real-time reverse transcriptase polymerase chain reaction at levels near adult values. Physiological measurements of the engineered skeletal muscle showed that they tetanize and display physiologic force length behavior, although developed force per cross-sectional area was below that of native rat skeletal muscle.

• Hyalomatrix: a temporary epidermal barrier, hyaluronan delivery, and neodermis induction system for keratinocyte stem cell therapy.

  Authors: Simon R Myers, Vaiude N Partha, Carlo Soranzo, Richard D Price, Harshad A Navsaria

  Tissue engineering. 13(11):2733-41.

  Keratinocyte stem cell technology provides at least an adjuvant therapy to clinically close large cutaneous wounds (e.g., burn wounds). Here, the performance of keratinocyte cultures dependsKeratinocyte stem cell technology provides at least an adjuvant therapy to clinically close large cutaneous wounds (e.g., burn wounds). Here, the performance of keratinocyte cultures depends primarily on the quality of the bed to which they are applied. Clinical take rates for cultured keratinocyte grafts are optimal when applied to a vascularized dermal bed with minimal bacterial colonization. In the absence of autologous dermis, staged reconstruction with a dermal equivalent or dermal regeneration template is required. A novel product, Hyalomatrix, is a bilayer of an esterified hyaluronan scaffold beneath a silicone membrane. The scaffold delivers hyaluronan to the wound bed, and the silicone membrane acts as a temporary epidermal barrier. The product has been investigated in a controlled, porcine, acute full-thickness excisional wound model. Cultured autologous keratinocytes (CAKs) were delivered on Laserskin to acute full-thickness wounds treated with Hyalomatrix within chambers, and graft take rates were assessed longitudinally using image analysis. In the absence of chambers, wound contraction was assessed. Clinical CAK take rates fall sequentially with delay in application post-Hyalomatrix pre-treatment, but repeated pre-treatment removed this, with maximal take of 57.2% at 5 weeks post-wounding. In the absence of chambers, more-complete wound closure resulted from edge re-epithelialization and contraction, by a factor of 5 at 1 month, and was achieved at least 2 weeks sooner in the gold standard controls of split-thickness autograft to an acute or pre-treated wound bed. Wound contraction and late neodermal morphology (1 year) were similar in pre-treated CAKs and split-thickness autograft wounds. In this model, the Hyalomatrix wound bed pre-treatment increase in CAK take appeared to be dose dependent. The product appeared to act as a hyaluronan delivery system rather than a dermal regeneration template. The silicone membrane may limit wound bed colonization, and the combination of this temporary barrier with hyaluronan delivery and neodermis induction has been termed a barrier-delivery-induction system. The development of similar systems for serial application offers an alternative to a dermal regeneration template when CAKs are engrafted in the hostile, colonized environment of large burn wounds.

• STRO-1 selected rat dental pulp stem cells transfected with adenoviral-mediated human bone morphogenetic protein 2 gene show enhanced odontogenic differentiation.

  Authors: Xuechao Yang, Peter M van der Kraan, Juliette van den Dolder, X Frank Walboomers, Zhuan Bian, Mingwen Fan, John A Jansen

  Tissue engineering. 13(11):2803-12.

  Dental pulp stem cells harbor great potential for tissue-engineering purposes. However, previous studies have shown variable results, and some have reported only limited osteogenic and odontogenicDental pulp stem cells harbor great potential for tissue-engineering purposes. However, previous studies have shown variable results, and some have reported only limited osteogenic and odontogenic potential.Because bone morphogenetic proteins (BMPs) are well-established agents to induce bone and dentin formation,in this study STRO-1-selected rat dental pulp-derived stem cells were transfected with the adenoviral mediated human BMP-2 gene. Subsequently, the cells were evaluated for their odontogenic differentiation ability in medium not containing dexamethasone or other stimuli. Cultures were investigated using light microscopy and scanning electron microscopy (SEM) and evaluated for cell proliferation, alkaline phosphatase(ALP) activity, and calcium content. Real-time polymerase chain reaction (PCR) was performed for gene expression of Alp, osteocalcin, collagen type I, bone sialoprotein, dentin sialophosphoprotein, and dentin matrix acidic phosphoprotein 1. Finally, an oligo-microarray was used to profile the expression of odontogenesis-related genes. Results of ALP activity, calcium content, and real-time PCR showed that only BMP2-transfected cells had the ability to differentiate into the odontoblast phenotype and to produce a calcified extracellular matrix. SEM and oligo-microarray confirmed these results. In contrast, the non-transfected cells represented a less differentiated cell phenotype. Based on our results, we concluded that the adenovirus can transfect STRO-1 selected cells with high efficacy. After BMP2 gene transfection, these cells had the ability to differentiate into odontoblast phenotype, even without the addition of odontogenic supplements to the medium.

• Cell guidance in tissue engineering: SDF-1 mediates site-directed homing of mesenchymal stem cells within three-dimensional polycaprolactone scaffolds.

  Authors: Jan-Thorsten Schantz, Harvey Chim, Matthew Whiteman

  Tissue engineering. 13(11):2615-24.

  Cell guidance is a new tissue engineering concept aimed at total in vivo tissue engineering without the need for cell seeding. This technique aims to create a biomimetic environment through constantCell guidance is a new tissue engineering concept aimed at total in vivo tissue engineering without the need for cell seeding. This technique aims to create a biomimetic environment through constant delivery of cytokines to different areas of an implanted scaffold, such that site-specific homing of cells can be achieved. In this study, expression of CXCR4 on mesenchymal stem cells (MSCs) was characterized by immunohistochemistry and flow cytometry, subsequent to which chemotaxis toward stromal cell-derived factor 1 (SDF-1) was demonstrated. In a subsequent three-dimensional in vitro study, MSCs were shown to migrate within a polycaprolactone scaffold in response to SDF-1, such that polarized tissue formation could be achieved. A customized cytokine microdelivery system comprising a reservoir housing system and microneedle apparatus was fabricated to ensure constant delivery of SDF-1 to the scaffold. Following on this experiment, we demonstrated in an in vivo rat bone tissue engineering model that a cytokine combination consisting of vascular endothelial growth factor, SDF-1, and bone morphogenetic protein-6 delivered at 10-day intervals through the microneedle apparatus could lead to tissue formation through migrating cell fronts, with evidence of angiogenesis and vascularization without the need for cell seeding on scaffolds prior to implantation. In summary, cell guidance offers an advancement in cellular methodology for tissue engineering, and promises a novel, minimally invasive option for tissue regeneration.

Data provided are for informational purposes only. Although carefully collected, accuracy cannot be guaranteed. The impact factor represents a rough estimation of the journal's impact factor and does not reflect the actual current impact factor. Publisher conditions are provided by RoMEO. Differing provisions from the publisher's actual policy or licence agreement may be applicable.