Journal of Tissue Engineering and Regenerative Medicine

Publications in this journal

• Article: Synthesis and characterization of poly(glycerol-co-sebacate-co-ε-caprolactone) elastomers.

  H M Aydin, K Salimi, M Yilmaz, M Turk, Z M O Rzayev, E Pişkin
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  ABSTRACT: In this study, poly(glycerol-co-sebacate-co-ε-caprolactone) (PGSCL) elastomers were synthesized for the first time from the respective monomers. The structural analysis of PGSCL elastomers by nuclear magnetic resonance ((1) H-NMR) and Fourier transform infrared spectroscopy (FTIR) revealed that the elastomers have a high number of hydrogen bonds and crosslinks. X-ray diffraction (XRD) and thermal analysis indicated an amorphous state. Differential scanning calorimetry (DSC) analysis showed that the elastomers has a glass transition temperature (Tg ) of -36.96°C. The Young's modulus and compression strength values were calculated as 46.08 MPa and 3.192 MPa, respectively. Calculations based on acid number and end groups analysis revealed a number average molecular weight of 148.15 kDa. Even though the foaming studies conducted by using supercritical CO2 resulted in a porous structure; the obtained morphology tended to disappear after 48 h, leaving small cracks on the surface. This phenomenon was interpreted as an indication of self-healing due to the high number of hydrogen bonds. The PGSCL elastomers synthesized in this study are flexible, robust to compression forces and have self-healing capacity. Thanks to good biocompatibility and poor cell-adhesion properties, the elastomers may find diverse applications where a postoperative adhesion barrier is required. Copyright © 2013 John Wiley & Sons, Ltd.
  Journal of Tissue Engineering and Regenerative Medicine 05/2013;
• Article: Plug and play: combining materials and technologies to improve bone regenerative strategies.

  Lorenzo Moroni, Anandkumar Nandakumar, Florence Barrère- de Groot, Clemens A van Blitterswijk, Pamela Habibovic
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  ABSTRACT: Despite recent advances in the development of biomaterials intended to replace natural bone grafts for the regeneration of large, clinically relevant defects, most synthetic solutions that are currently applied in the clinic are still inferior to natural bone grafts with regard to regenerative potential and are limited to non-weight-bearing applications. From a materials science perspective, we always face the conundrum of the preservation of bioactivity of calcium phosphate ceramics in spite of better mechanical and handling properties and processability of polymers. Composites have long been investigated as a method to marry these critical properties for the successful regeneration of bone and, indeed, have shown a significant improvement when used in combination with cells or growth factors. However, when looking at this approach from a clinical and regulatory perspective, the use of cells or biologicals prolongs the path of new treatments from the bench to the bedside. Applying 'smart' synthetic materials alone poses the fascinating challenge of instructing tissue regeneration in situ, thereby tremendously facilitating clinical translation. In the journey to make this possible, and with the aim of adding up the advantages of different biomaterials, combinations of fabrication technologies arise as a new strategy for generating instructive three-dimensional (3D) constructs for bone regeneration. Here we provide a review of recent technologies and approaches to create such constructs and give our perspective on how combinations of technologies and materials can help in obtaining more functional bone regeneration. Copyright © 2013 John Wiley & Sons, Ltd.
  Journal of Tissue Engineering and Regenerative Medicine 05/2013;
• Article: Cell-cell interaction between vocal fold fibroblasts and bone marrow mesenchymal stromal cells in three-dimensional hyaluronan hydrogel.

  Xia Chen, Susan L Thibeault
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  ABSTRACT: Mesenchymal stromal cells (MSCs) are multipotential adult cells present in all tissues. Paracrine effects and differentiating ability make MSCs an ideal cell source for tissue regeneration. However, little is known about how interactions between implanted MSCs and native cells influence cellular growth, proliferation, and behaviour. By using an in vitro three-dimensional (3D) co-culture assay of normal or scarred human vocal fold fibroblasts (VFFs) and bone marrow-derived MSCs (BM-MSCs) in a uniquely suited hyaluronan hydrogel (HyStem-VF), we investigated cell morphology, survival rate, proliferation and protein and gene expression of VFFs and BM-MSCs. BM-MSCs inhibited cell proliferation of both normal and scarred VFFs without changes in VFF morphology or viability. BM-MSCs demonstrated decreased proliferation and survival rate after 7 days of co-culture with VFFs. Interactions between BM-MSCs and VFFs led to a significant increase in protein secretion of collagen I and hepatocyte growth factor (HGF) and a decrease of vascular endothelial growth factor (VEGF), monocyte chemotactic protein-1 (MCP-1) and interleukin-6 (IL-6). In particular, BM-MSCs significantly upregulated matrix metalloproteinase 1 (MMP1) and HGF gene expression for scarred VFFs compared to normal VFFs, indicating the potential for increases in extracellular matrix remodelling and tissue regeneration. Application of BM-MSCs-hydrogels may play a significant role in tissue regeneration, providing a therapeutic approach for vocal fold scarring. Copyright © 2013 John Wiley & Sons, Ltd.
  Journal of Tissue Engineering and Regenerative Medicine 05/2013;
• Article: T cell subsets differently regulate osteogenic differentiation of human mesenchymal stromal cells in vitro.

  Francesco Grassi, Luca Cattini, Laura Gambari, Cristina Manferdini, Anna Piacentini, Elena Gabusi, Andrea Facchini, Gina Lisignoli
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  ABSTRACT: T lymphocytes play a key role in the regulation of bone homeostasis and bone healing. The inflammatory response at the site of bone injury is essential to the initiation of the bone repair program; however, an uncontrolled exposure to inflammatory environment has a negative effect on tissue regeneration - indeed, activated T cells were shown to inhibit osteogenic differentiation on human mesenchymal stromal cells (MSCs). Whether resting T cells can induce osteogenic differentiation of MSCs and what role specific T cells subset play in this process is still elusive. In this study, we sought to analyse the osteogenic gene expression profile of whole T cells, CD4 and CD8 T cells isolated from healthy donors and investigated whether secreted factors from each group modulate osteogenic differentiation of human MSCs. Gene expression profiling identified a pool of 51 genes involved at various stages in bone growth which are expressed above detectable levels in CD4 and CD8 T cells. Most genes of this pool were expressed at higher levels in the CD4 subset. In vitro mineralization assays revealed that conditioned medium from CD4 T cells, but not from CD8 cells, significantly increased mineralization in osteogenic cultures of human MSCs; furthermore, mRNA expression of Runt-related transcription factor 2 (RUNX-2), osteocalcin (OC), bone sialoprotein (BSP) and alkaline phosphatase (ALP) in MSCs was significantly upregulated in the presence of CD4-conditioned medium but not with that obtained from CD8. The results show a differential role for CD4 and CD8 T cells in supporting bone formation and identify an osteogenic gene signature of each subset. Copyright © 2013 John Wiley & Sons, Ltd.
  Journal of Tissue Engineering and Regenerative Medicine 05/2013;
• Article: Effectiveness of hybridized nano- and microstructure biodegradable, biocompatible, collagen-based, three-dimensional bioimplants in repair of a large tendon-defect model in rabbits.

  Ali Moshiri, Ahmad Oryan, Abdulhamid Meimandi-Parizi, Ian A Silver, Nader Tanideh, Navid Golestani
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  ABSTRACT: This study was designed to investigate the effectiveness of hybridized, three-dimensional (3D) collagen implants in repair of experimentally-induced tendon defects in rabbits. Seventy-five mature New Zealand albino rabbits were divided into treated (n = 50) and control (n = 20) groups. The left Achilles tendon was completely transected and 2 cm excised. In treated animals defects were filled with hybridized collagen implants and repaired with sutures. In control rabbits tendon defects were sutured similarly but the gap was left untreated. Changes in injured and normal contralateral tendons were assessed weekly by ultrasonography. Among the treated animals, small pilot groups were euthanized at 5, 10, 15, 20, 30, 40 (n = 5 at each time interval) and the remainder (n = 20) at 60 days post-injury. All control animals were euthanized at 60 days. Tendon lesions of all animals were examined morphologically and histologically immediately after death. Those of the experimental groups (n = 20 for each) were examined for gross pathological, histopathological and ultrastructural changes together with dry matter content at 60 days post-injury, as were the normal, contralateral tendons of both groups. In comparison with healing lesions of control animals, the treated tendons showed greater numbers of mature tenoblasts and tenocytes, minimal peritendinous adhesions and oedema, together with greater echogenicity, homogeneity and fibril alignment. Fewer chronic inflammatory cells were present in treated than control tendons. Hybridized collagen implants acted as scaffolds for tenoblasts and longitudinally-orientated newly-formed collagen fibrils, which encouraged tendon repair with homogeneous, well-organized highly aligned scar tissue that was histologically and ultrastructurally more mature than in untreated controls. Copyright © 2013 John Wiley & Sons, Ltd.
  Journal of Tissue Engineering and Regenerative Medicine 05/2013;
• Article: The evolution of simulation techniques for dynamic bone tissue engineering in bioreactors.

  Jolanda Rita Vetsch, Ralph Müller, Sandra Hofmann
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  ABSTRACT: Bone tissue engineering aims to overcome the drawbacks of current bone regeneration techniques in orthopaedics. Bioreactors are widely used in the field of bone tissue engineering, as they help support efficient nutrition of cultured cells with the possible combination of applying mechanical stimuli. Beneficial influencing parameters of in vitro cultures are difficult to find and are mostly determined by trial and error, which is associated with significant time and money spent. Mathematical simulations can support the finding of optimal parameters. Simulations have evolved over the last 20 years from simple analytical models to complex and detailed computational models. They allow researchers to simulate the mechanical as well as the biological environment experienced by cells seeded on scaffolds in a bioreactor. Based on the simulation results, it is possible to give recommendations about specific parameters for bone bioreactor cultures, such as scaffold geometries, scaffold mechanical properties, the level of applied mechanical loading or nutrient concentrations. This article reviews the evolution in simulating various aspects of dynamic bone culture in bioreactors and reveals future research directions. Copyright © 2013 John Wiley & Sons, Ltd.
  Journal of Tissue Engineering and Regenerative Medicine 04/2013;
• Article: Carnitine and acetylcarnitine modulate mesenchymal differentiation of adult stem cells.

  Qiaozhi Lu, Yuanfan Zhang, Jennifer H Elisseeff
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  ABSTRACT: Cellular metabolic activity, especially mitochondrial metabolism, plays a vital role in regulating cell proliferation and differentiation. Metabolism could therefore be an important factor to consider when using engineering technologies to stimulate tissue development and repair. The small metabolite carnitine and its derivative acetylcarnitine affect the activities of several pathways in mitochondrial metabolism, but their influence on cell differentiation has not yet been thoroughly studied. To elucidate the effects of these two small molecules on mesenchymal tissue engineering, we used adult stem cells as a platform in both monolayer and 3D hydrogel culture systems. We investigated the impact of these two small molecules on the differentiation of adult stem cells and analysed gene expression, cell proliferation and extracellular matrix deposition. We found that the molecules reduced adipogenesis but stimulated osteogenesis and chondrogenesis in both culture systems. Our results suggest that carnitine and acetylcarnitine could affect the differentiation rate of adult stem cells by regulating mitochondrial metabolism. The effects of these two small molecules give rise to the possibility of employing such metabolites in tissue-engineering systems to enhance cell differentiation and tissue development. Copyright © 2013 John Wiley & Sons, Ltd.
  Journal of Tissue Engineering and Regenerative Medicine 04/2013;
• Article: Ameloblasts serum-free conditioned medium: bone morphogenic protein 4-induced odontogenic differentiation of mouse induced pluripotent stem cells.

  Li Liu, Ying-Feng Liu, Jing Zhang, Yin-Zhong Duan, Yan Jin
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  ABSTRACT: Induced pluripotent stem (iPS) cells possess the ability of self-renewal and can differentiate into cells of the three germ layers, both in vitro and in vivo. Here we report a new method to efficiently induce differentiation of mouse iPS cells into the odontogenic lineage. Using ameloblasts serum-free conditioned medium (ASF-CM), we successfully generated ameloblast-like cells from mouse iPS cells. Importantly, culturing mouse iPS cells in ASF-CM supplemented with BMP4 (ASF-BMP4) promoted odontogenic differentiation, which was evident by the upregulation of ameloblast-specific as well as odontoblast-specific genes. On the other hand, culturing mouse iPS cells in ASF-CM supplemented with noggin (ASF-noggin), an inhibitor of BMP4, abrogated this effect. These results suggest that mouse iPS cells can be induced by ASF-BMP4 to differentiate into ameloblast-like and odontoblast-like cells. The results of our study raise the possibility of using patient-specific iPS cells for tooth regeneration in the future. Copyright © 2013 John Wiley & Sons, Ltd.
  Journal of Tissue Engineering and Regenerative Medicine 04/2013;
• Article: Iron dose-dependent differentiation and enucleation of human erythroblasts in serum-free medium.

  Colleen Byrnes, Y Terry Lee, Emily R Meier, Antoinette Rabel, David B Sacks, Jeffery L Miller
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  ABSTRACT: Improvements in ex vivo generation of enucleated red blood cells are being sought for erythroid biology research, toward the ultimate goal of erythrocyte engineering for clinical use. Based upon the high levels of iron-saturated transferrin in plasma serum, it was hypothesized that terminal differentiation in serum-free media may be highly dependent on the concentration of iron. Here adult human CD34(+) cells were cultured in a serum-free medium containing dosed levels of iron-saturated transferrin (holo-Tf, 0.1-1.0 mg/ml). Iron in the culture medium was reduced, but not depleted, with erythroblast differentiation into haemoglobinized cells. At the lowest holo-Tf dose (0.1 mg/ml), terminal differentiation was significantly reduced and the majority of the cells underwent apoptotic death. Cell survival, differentiation and enucleation were enhanced as the holo-Tf dose increased. These data suggest that adequate holo-Tf dosing is critical for terminal differentiation and enucleation of human erythroblasts generated ex vivo in serum-free culture conditions. Published 2013. This article is a US Government work and is in the public domain in the USA.
  Journal of Tissue Engineering and Regenerative Medicine 04/2013;
• Article: Three-dimensional assembly of tissue-engineered cartilage constructs results in cartilaginous tissue formation without retainment of zonal characteristics.

  W Schuurman, E B Harimulyo, D Gawlitta, T B F Woodfield, W J A Dhert, P R van Weeren, J Malda
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  ABSTRACT: Articular cartilage has limited regenerative capabilities. Chondrocytes from different layers of cartilage have specific properties, and regenerative approaches using zonal chondrocytes may yield better replication of the architecture of native cartilage than when using a single cell population. To obtain high seeding efficiency while still mimicking zonal architecture, cell pellets of expanded deep zone and superficial zone equine chondrocytes were seeded and cultured in two layers on poly(ethylene glycol)-terephthalate-poly(butylene terephthalate) (PEGT-PBT) scaffolds. Scaffolds seeded with cell pellets consisting of a 1:1 mixture of both cell sources served as controls. Parallel to this, pellets of superficial or deep zone chondrocytes, and combinations of the two cell populations, were cultured without the scaffold. Pellet cultures of zonal chondrocytes in scaffolds resulted in a high seeding efficiency and abundant cartilaginous tissue formation, containing collagen type II and glycosaminoglycans (GAGs) in all groups, irrespective of the donor (n = 3), zonal population or stratified scaffold-seeding approach used. However, whereas total GAG production was similar, the constructs retained significantly more GAG compared to pellet cultures, in which a high percentage of the produced GAGs were secreted into the culture medium. Immunohistochemistry for zonal markers did not show any differences between the conditions. We conclude that spatially defined pellet culture in 3D scaffolds is associated with high seeding efficiency and supports cartilaginous tissue formation, but did not result in the maintenance or restoration of the original zonal phenotype. The use of pellet-assembled constructs leads to a better retainment of newly produced GAGs than the use of pellet cultures alone. Copyright © 2013 John Wiley & Sons, Ltd.
  Journal of Tissue Engineering and Regenerative Medicine 04/2013;
• Article: Ultrasound-mediated gene transfer (sonoporation) in fibrin-based matrices: potential for use in tissue regeneration.

  Nikolitsa Nomikou, Georg A Feichtinger, Heinz Redl, Anthony P McHale
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  ABSTRACT: It has been suggested that gene transfer into donor cells is an efficient and practical means of locally supplying requisite growth factors for applications in tissue regeneration. Here we describe, for the first time, an ultrasound-mediated system that can non-invasively facilitate gene transfer into cells entrapped within fibrin-based matrices. Since ultrasound-mediated gene transfer is enhanced using microbubbles, we compared the efficacy of neutral and cationic forms of these reagents on the ultrasound-stimulated gene transfer process in gel matrices. In doing so we demonstrated the beneficial effects associated with the use of cationic microbubble preparations that interact directly with cells and nucleic acid within matrices. In some cases, gene expression was increased two-fold in gel matrices when cationic microbubbles were compared with neutral microbubbles. In addition, incorporating collagen into fibrin gels yielded a 25-fold increase in gene expression after application of ultrasound to microbubble-containing matrices. We suggest that this novel system may facilitate non-invasive temporal and spatial control of gene transfer in gel-based matrices for the purposes of tissue regeneration. Copyright © 2013 John Wiley & Sons, Ltd.
  Journal of Tissue Engineering and Regenerative Medicine 04/2013;
• Article: Ex vivo expansion of cord blood haematopoietic stem/progenitor cells under physiological oxygen tensions: clear-cut effects on cell proliferation, differentiation and metabolism.

  Pedro Z Andrade, António M de Soure, Francisco Dos Santos, Artur Paiva, Joaquim M S Cabral, Cláudia L da Silva
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  ABSTRACT: Physiologically low O2 tensions are believed to regulate haematopoietic stem cell (HSC) functions in the bone marrow (BM; 0-5%). In turn, placenta and umbilical cord are characterized by slightly higher physiological O2 tensions (3-10%). We hypothesized that O2 concentrations within this range may be exploited to augment the ex vivo expansion/maintenance of HSCs from umbilical cord (placental) blood (UCB). The expansion of UCB CD34(+) -enriched cells was studied in co-culture with BM mesenchymal stem/stromal cells (MSCs) under 2%, 5%, 10% and 21% O2 . 2% O2 resulted in a significantly lower CD34(+) cell expansion (25-fold vs 60-, 64- and 92-fold at day 10 for 5%, 21%, 10% O2 , respectively). In turn, 10% O2 promoted the highest CD34(+) CD90(+) cell expansion, reaching 22 ± 5.4- vs 5.6 ± 2.4- and 5.7 ± 2.0-fold for 2%, 5% and 21% O2 , respectively, after 14 days. Similar differentiation patterns were observed under different O2 tensions, being primarily shifted towards the neutrophil lineage. Cell division kinetics revealed a higher proliferative status of cells cultured under 10% and 21% vs 2% O2 . Expectedly, higher specific glucose consumption and lactate production rates were determined at 2% O2 when compared to higher O2 concentrations (5-21%). Overall, these results suggest that physiological oxygen tensions, in particular 10% O2 , can maximize the ex vivo expansion of UCB stem/progenitor cells in co-culture with BM MSCs. Importantly, these studies highlight the importance of exploiting knowledge of the intricate microenvironment of the haematopoietic niche towards the definition of efficient and controlled ex vivo culture systems capable of generating large HSCs numbers for clinical applications. Copyright © 2013 John Wiley & Sons, Ltd.
  Journal of Tissue Engineering and Regenerative Medicine 04/2013;
• Article: Silk fibroin scaffolds enhance cell commitment of adult rat cardiac progenitor cells.

  Valentina Di Felice, Claudia Serradifalco, Luigi Rizzuto, Angela De Luca, Francesca Rappa, Rosario Barone, Patrizia Di Marco, Giovanni Cassata, Roberto Puleio, Lucia Verin, Antonella Motta, Claudio Migliaresi, Annalisa Guercio, Giovanni Zummo
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  ABSTRACT: The use of three-dimensional (3D) cultures may induce cardiac progenitor cells to synthesize their own extracellular matrix (ECM) and sarcomeric proteins to initiate cardiac differentiation. 3D cultures grown on synthetic scaffolds may favour the implantation and survival of stem cells for cell therapy when pharmacological therapies are not efficient in curing cardiovascular diseases and when organ transplantation remains the only treatment able to rescue the patient's life. Silk fibroin-based scaffolds may be used to increase cell affinity to biomaterials and may be chemically modified to improve cell adhesion. In the present study, porous, partially orientated and electrospun nanometric nets were used. Cardiac progenitor cells isolated from adult rats were seeded by capillarity in the 3D structures and cultured inside inserts for 21 days. Under this condition, the cells expressed a high level of sarcomeric and cardiac proteins and synthesized a great quantity of ECM. In particular, partially orientated scaffolds induced the synthesis of titin, which is a fundamental protein in sarcomere assembly. Copyright © 2013 John Wiley & Sons, Ltd.
  Journal of Tissue Engineering and Regenerative Medicine 04/2013;
• Article: Collagen modules for in situ delivery of mesenchymal stromal cell-derived endothelial cells for improved angiogenesis.

  Karolina Janeczek Portalska, M Dean Chamberlain, Chuen Lo, Clemens van Blitterswijk, Michael V Sefton, Jan de Boer
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  ABSTRACT: Modular tissue engineering is a strategy to create scalable, self-assembling, three-dimensional (3D) tissue constructs. This strategy was used to deliver endothelial-like cells derived from bone marrow mesenchymal stromal cells (EL-MSCs) to locally induce vascularization. First, tissue engineered modules were formed, comprising EL-MSCs and collagen-based cylinders. Seven days of module culture in a microfluidic chamber under continuous flow resulted in the formation of interstices, formed by random packing of the modules, which served as channels and were lined by the EL-MSCs. We observed maintenance of the endothelial phenotype of the EL-MSCs, as demonstrated by CD31 staining, and the cells proliferated well. Next, collagen modules covered with EL-MSCs, with or without embedded MSCs, were implanted subcutaneously in immune-compromised SCID/Bg mice. After 7 days, CD31-positive vessels were observed in the samples. These data demonstrate the feasibility of EL-MSCs coated collagen module as a strategy to locally stimulate angiogenesis and vasculogenesis. Copyright © 2013 John Wiley & Sons, Ltd.
  Journal of Tissue Engineering and Regenerative Medicine 04/2013;
• Article: Synthesis and physicochemical, in vitro and in vivo evaluation of an anisotropic, nanocrystalline hydroxyapatite bisque scaffold with parallel-aligned pores mimicking the microstructure of cortical bone.

  F Despang, A Bernhardt, A Lode, R Dittrich, T Hanke, S J Shenoy, S Mani, A John, M Gelinsky
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  ABSTRACT: Scaffolds for bone regeneration are mostly prepared with an isotropic, sponge-like structure mimicking the architecture of trabecular bone. We have developed an anisotropic bioceramic with parallel aligned pores resembling the honeycomb arrangement of Haversian canals of cortical bone and investigated its potential as a scaffold for tissue engineering. Parallel channel-like pores were generated by ionotropic gelation of an alginate-hydroxyapatite (HA) slurry, followed by ceramic processing. Organic components were thermally removed at 650 °C, whereas the pore system was preserved in the obtained HA bioceramic in the processing stage of a bisque. Even without further sintering at higher temperatures, the anisotropic HA bisque (AHAB) became mechanically stable with a compressive strength (4.3 MPa) comparable to that of native trabecular bone. Owing to the low-temperature treatment, a nanocrystalline microstructure with high porosity (82%) and surface area (24.9 m(2) /g) was achieved that kept the material dissolvable in acidic conditions, similar to osteoclastic degradation of bone. Human mesenchymal stem cells (hMSCs) adhered, proliferated and differentiated into osteoblasts when osteogenically induced, indicating the cytocompatibility of the bisque scaffold. Furthermore, we demonstrated fusion of human monocytes to osteoclast-like cells in vitro on this substrate, similar to the natural pathway. Biocompatibility was demonstrated in vivo by implantation of the bisque ceramic into cortical rabbit femur defects, followed by histological analysis, where new bone formation inside the channel-like pores and generation of an osteon-like tissue morphology was observed. Copyright © 2013 John Wiley & Sons, Ltd.
  Journal of Tissue Engineering and Regenerative Medicine 04/2013;
• Article: Development of a morphogenetically active scaffold for three-dimensional growth of bone cells: biosilica-alginate hydrogel for SaOS-2 cell cultivation.

  Werner E G Müller, Heinz C Schröder, Qingling Feng, Ute Schlossmacher, Thorben Link, Xiaohong Wang
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  ABSTRACT: Polymeric silica is formed from ortho-silicate during a sol-gel formation process, while biosilica is the product of an enzymatically driven bio-polycondensation reaction. Both polymers have recently been described as a template that induces an increased expression of the genes encoding bone morphogenetic protein 2 (BMP-2) and osteoprotegerin in osteoblast-related SaOS-2 cells; simultaneously or subsequently the cells respond with enhanced hydroxyapatite formation. In order to assess whether the biocompatible polymeric silica/biosilica can serve as a morphogenetically active matrix suitable for three-dimensional (3D) cell growth, or even for 3D cell bioprinting, SaOS-2 cells were embedded into a Na-alginate-based hydrogel. Four different gelatinous hydrogel matrices were used for suspending SaOS-2 cells: (a) the hydrogel alone; (b) the hydrogel with 400 μm ortho-silicate; (c) the hydrogel supplemented with 400 μm ortho-silicate and recombinant silicatein to allow biosilica synthesis to occur; and (d) the hydrogel with ortho-silicate and BSA. The SaOS-2 cells showed an increased growth if silica/biosilica components were present in the hydrogel. Likewise intensified was the formation of hydroxyapatite nodules in the silica-containing hydrogels. After an incubation period of 2 weeks, cells present in silica-containing hydrogels showed a significantly higher expression of the genes encoding the cytokine BMP-2, the major fibrillar structural protein collagen 1 and likewise of carbonic anhydrase. It is concluded that silica, and to a larger extent biosilica, retains its morphogenetic/osteogenic potential after addition to Na-alginate-based hydrogels. This property might qualify silica hydrogels to be also used as a matrix for 3D cell printing. Copyright © 2013 John Wiley & Sons, Ltd.
  Journal of Tissue Engineering and Regenerative Medicine 04/2013;
• Article: FGF, TGFβ and Wnt crosstalk: embryonic to in vitro cartilage development from mesenchymal stem cells.

  Mairéad A Cleary, Gerjo J V M van Osch, Pieter A Brama, Catharine A Hellingman, Roberto Narcisi
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  ABSTRACT: Articular cartilage is easily damaged, yet difficult to repair. Cartilage tissue engineering seems a promising therapeutic solution to restore articular cartilage structure and function, with mesenchymal stem cells (MSCs) receiving increasing attention for their promise to promote cartilage repair. It is known from embryology that members of the fibroblast growth factor (FGF), transforming growth factor-β (TGFβ) and wingless-type (Wnt) protein families are involved in controlling different differentiation stages during chondrogenesis. Individually, these pathways have been extensively studied but so far attempts to recapitulate embryonic development in in vitro MSC chondrogenesis have failed to produce stable and functioning articular cartilage; instead, transient hypertrophic cartilage is obtained. We believe a better understanding of the simultaneous integration of these factors will improve how we relate embryonic chondrogenesis to in vitro MSC chondrogenesis. This narrative review attempts to define current knowledge on the crosstalk between the FGF, TGFβ and Wnt signalling pathways during different stages of mesenchymal chondrogenesis. Connecting embryogenesis and in vitro differentiation of human MSCs might provide insights into how to improve and progress cartilage tissue engineering for the future. Copyright © 2013 John Wiley & Sons, Ltd.
  Journal of Tissue Engineering and Regenerative Medicine 04/2013;
• Article: Adipose-derived stromal cells for the reconstruction of a human vesical equivalent.

  Alexandre Rousseau, Julie Fradette, Geneviève Bernard, Robert Gauvin, Véronique Laterreur, Stéphane Bolduc
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  ABSTRACT: Despite a wide panel of tissue-engineering models available for vesical reconstruction, the lack of a differentiated urothelium remains their main common limitation. For the first time to our knowledge, an entirely human vesical equivalent, free of exogenous matrix, has been reconstructed using the self-assembly method. Moreover, we tested the contribution of adipose-derived stromal cells, an easily available source of mesenchymal cells featuring many potential advantages, by reconstructing three types of equivalent, named fibroblast vesical equivalent, adipose-derived stromal cell vesical equivalent and hybrid vesical equivalent - the latter containing both adipose-derived stromal cells and fibroblasts. The new substitutes have been compared and characterized for matrix composition and organization, functionality and mechanical behaviour. Although all three vesical equivalents displayed adequate collagen type I and III expression, only two of them, fibroblast vesical equivalent and hybrid vesical equivalent, sustained the development of a differentiated and functional urothelium. The presence of uroplakins Ib, II and III and the tight junction marker ZO-1 was detected and correlated with impermeability. The mechanical resistance of these tissues was sufficient for use by surgeons. We present here in vitro tissue-engineered vesical equivalents, built without the use of any exogenous matrix, able to sustain mechanical stress and to support the formation of a functional urothelium, i.e. able to display a barrier function similar to that of native tissue. Copyright © 2013 John Wiley & Sons, Ltd.
  Journal of Tissue Engineering and Regenerative Medicine 04/2013;
• Article: The application of POSS nanostructures in cartilage tissue engineering: the chondrocyte response to nanoscale geometry.

  Adelola O Oseni, Peter E Butler, Alexander M Seifalian
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  ABSTRACT: Despite extensive research into cartilage tissue engineering (CTE), there is still no scaffold ideal for clinical applications. Various synthetic and natural polymers have been investigated in vitro and in vivo, but none have reached widespread clinical use. The authors investigate the potential of POSS-PCU, a synthetic nanocomposite polymer, for use in CTE. POSS-PCU is modified with silsesquioxane nanostructures that improve its biological and physical properties. The ability of POSS-PCU to support the growth of ovine nasoseptal chondrocytes was evaluated against a polymer widely used in CTE, polycaprolactone (PCL). Scaffolds with varied concentrations of the POSS molecule were also synthesized to investigate their effect on chondrocyte growth. Chondrocytes were seeded onto scaffold disks (PCU negative control; POSS-PCU 2%, 4%, 6%, 8%; PCL). Cytocompatibilty was evaluated using cell viability, total DNA, collagen and GAG assays. Chondrocytes cultured on POSS-PCU (2% POSS) scaffolds had significantly higher viability than PCL scaffolds (p < 0.001). Total DNA, collagen and sGAG protein were also greater on POSS-PCU scaffolds compared with PCL (p > 0.05). POSS-PCU (6% and 8% POSS) had improved viability and proliferation over an 18 day culture period compared with 2% and 4% POSS-PCU (p < 0.0001). Increasing the percentage of POSS in the scaffolds increased the size of the pores found in the scaffolds (p < 0.05). POSS-PCU has excellent potential for use in CTE. It supports the growth of chondrocytes in vitro and the POSS modification significantly enhances the growth and proliferation of nasoseptal chondrocytes compared with traditional scaffolds such as PCL. Copyright © 2013 John Wiley & Sons, Ltd.
  Journal of Tissue Engineering and Regenerative Medicine 04/2013;
• Article: An in vitro expansion score for tissue-engineering applications with human bone marrow-derived mesenchymal stem cells.

  Alessandro Bertolo, Marco Mehr, Tiziana Janner-Jametti, Ursula Graumann, Niklaus Aebli, Martin Baur, Stephen J Ferguson, Jivko V Stoyanov
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  ABSTRACT: Human bone marrow-derived mesenchymal stem cells (MSCs) have limited growth potential in vitro and cease to divide due to replicative senescence, which from a tissue-engineering perspective has practical implications, such as defining the correct starting points for differentiation and transplantation. Time spent in culture before the loss of required differentiation potential is different and reflects patient variability, which is a problem for cell expansion. This study aimed to develop a score set which can be used to quantify the senescent state of MSCs and predict whether cells preserve their ability to differentiate to osteogenic, adipogenic and chondrogenic phenotypes, based on colony-forming unit (CFU) assay, population doubling time (PDT), senescence-associated β-galactosidase (SA-β-Gal) activity, cell size, telomere length and gene expression of MSCs cultured in vitro over 11 passages. This set of morphological, physiological and genetic senescence markers was correlated to the ability of MSCs to differentiate. Differentiation efficiency was assessed by marker genes and protein expression. CFUs decreased with increasing passage number, whereas SA-β-Gal activity and PDT increased; however, the correlation with MSCs' differentiation potential was sometimes unexpected. The expression of genes related to senescence was higher in late-passage cells than in early-passage cells. Early-passage cells underwent efficient osteogenic differentiation, with mid-passage cells performing best in chondrogenic differentiation. Late-passage cells preserve only adipogenic differentiation potential. Based on this marker set, we propose a senescence score in which combined markers give a reliable quality control of MSCs, not depending only on mechanistic passage number. Copyright © 2013 John Wiley & Sons, Ltd.
  Journal of Tissue Engineering and Regenerative Medicine 04/2013;