Weiss EC, Anastasiadis P, Pilarczyk G, Lemor RM, Zinin PV. ...... of the cord with the calculation of the stromal- vascular rate (SVR) using simple formula.
Keynote Lecture
KL-001
Differentiation and conversion of human cell fate: plasticity and pluripotency *M Bhatia1 1
McMaster University, Stem Cell and Cancer Research Institute (SCC-RI), Hamilton, ON, Canada
The ability to reprogram human skin cells to pluripotent stem cell fate, akin to embryonic stem cells, has transformed the views, approaches and possibilities toward applications in regenerative medicine. However, differentiation into cell types required for drug discovery or cell-replacement therapies from these pluripotent stem cells remains a major limitation. Recent advancements in cell reprogramming beyond pluripotency, and use of different methods to retaining pluripotency in vitro from unique sources of human tissue may circumvent these limitations in differentiation. These recent advancements of cell fate conversion and their practical utility will be discussed.
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1
Keynote Lecture
KL-002
Biological parameters determining the clinical outcome of autologous cultures of limbal stem cells *M De Luca1 University of Modena and Reggio Emilia, Centre for Regenerative Medicine “Stefano Ferrari”, Modena, Italy
1
Objectives Limbal cultures restore the corneal epithelium in patients with ocular burns. We investigate biological parameters instrumental for their clinical success. Materials & methods We report a long-term multicenter prospective study on 152 patients, carrying severe burn-dependent corneal destruction, treated with autologous limbal cells cultured on fibrin and clinical-grade 3T3–J2 feeder cells. Clinical results were statistically evaluated both by parametric and non-parametric methods. Clinical outcomes were scored as full success, partial success and failure in 66.05, 19.14, and 14.81% of eyes, respectively. Total number of clonogenic cells, colony size, growth rate and presence of conjunctival cells could not predict clinical results. Instead, clinical data provided conclusive evidence that graft quality and likelihood of a successful outcome rely on an accurate evaluation of the number of stem cells detected before transplantation as holoclones expressing high levels of the p63 transcription factor. Results No adverse effects related to the feeder-layer has been observed and the regenerated epithelium was completely devoid of any 3T3 contamination. Cultures of limbal stem cells can be safely used to successfully treat massive destruction of the human cornea. Conclusion We emphasize the importance of a discipline for defining the suitability and the quality of cultured epithelial grafts, which are relevant to the future clinical use of any cultured cell type.
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2
Keynote Lecture
KL-003
Multifunctional biomaterials *A Lendlein1 1
Helmholtz-Zentrum Geesthacht, Institute of Biomaterial Science, Teltow, Germany
Polymers are applied today in a variety of clinical applications. Interestingly, most of these materials were initially not developed for medicine, but were available as engineering plastics for industrial applications. The success of polymerbased medical devices and controlled drug delivery systems has motivated many proposals for novel potential applications. Each application demands a specific combination of biomaterial’s properties and functions. With a growing number of applications the number of materials required differing substantially in their property/function profile is increasing as well. Therefore, polymer systems are required, which allow to tailor properties and functions. The design of multifunctional biomaterials is exemplarily illustrated for cardiovascular implants, such as degradable stents and devices for minimally invasive surgery. Finally, the potential of polymer-based biomaterials in regenerative therapies is outlined. Information resources 1
Jung F, Wischke C, Lendlein A. Degradable, multifunctional cardiovascular implants: challenges and hurdles. MRS Bull. 35, 607–613 (2010).
2
Shastri P, Lendlein A. Materials in regenerative medicine. Adv. Mater. 21, 3231–3234 (2009).
3
Behl M, Razzaq M, Lendlein A. Multifunctional shape-memory polymers. Adv. Mater. 22, 3388–3410 (2010).
4
Lendlein A, Behl M, Hiebl B, Wischke C. Shape-memory polymers as a technology platform for biomedical applications. Expert Rev. Med. Dev. 7, 357–379 (2010).
5
Wischke C, Krüger A, Roch T et al. Endothelial cell response to (co)polymer nanoparticles depending on the inflammatory environment and comonomer ratio. Europ. J. Pharm. Biopharm. 84, 288–296 (2013).
6
Wischke C, Behl M, Lendlein A. Drug-releasing shape-memory polymers - the role of morphology, processing effects, and matrix degradation. Expert Opin. Drug Del. 10(9), 1193–1205 (2013).
7
Sauter T, Kratz K, Lendlein A. Pore-size distribution controls shape-memory properties on the macro- and microscale of polymeric foams. Macromol. Chem. Phys. 214, 1184–1188 (2013).
8
Lendlein A, Langer R. Biodegradable, elastic shape-memory polymers for potential biomedical applications. Science 296, 1673–1676 (2002).
9
Kratz K, Voigt U, Lendlein A. Temperature-memory effect of copolyesterurethanes and their application potential in minimally invasive medical technologies. Adv. Funct. Mater. 22, 3057–3065 (2012).
10 Behl M, Kratz K, Zotzmann J, Nöchel U, Lendlein A. Reversible bidirectional shape-memory polymers. Adv. Mater. 25(32),4466–4469
(2013).
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3
Keynote Lecture
KL-004
Umbilical cord blood-derived neural stem cells and their potential use for ischemic brain damage *B Lukomska1 1
Mossakowski Medical Research Centre, NeuroRepair Department, Warsaw, Poland
Several stem cell-based therapeutic tools are being investigated for regeneration of CNS injuries and neurodegenerative diseases. In contrast to the majority of cells isolated from adult tissues, stem cells (SC) in umbilical cord and cord blood (CB) possess several unique characteristics. The main drawback of CB is the limited number of cells available from the single unit. Therefore, the development of methods of direct expansion of certain lineages according to therapeutic demands is the main goal of studies. In our study we evaluated culture conditions to obtain CB progenitor cells exhibiting neurogenic potential in vitro. The assessment of different culture conditions revealed that dense cell culture, which enables spherical aggregates of cells floating in serum free-media under constant rotary movement, is the optimal circumstance for CB cells to obtain neurally committed progenitors with high proliferation potential and slower rate of differentiation. Experimental criteria obtained by our group and long-term experience with neuronal commitment of CB cells provided the basis for clinical therapy. Together with clinicians we transplanted intracerebroventricularly autologous neurally committed cord blood cells (CB-NPs) labeled by SPIO nanoparticles in a child with global brain ischemia injury. MRI examination performed after administration of CB-NPs to the right cerebral hemisphere disclosed SPIO-tagged cells persistent in the injecting site up to 3 months. The results of the study indicated that intracerebroventricular transplantation of autologous neurally committed CB cells is feasible, safe and well tolerated and labeled by SPIO could be noninvasible monitored.
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4
Keynote Lecture
KL-005
Cardiomyocytes from human pluripotent stem cells: the new patient in safety pharmacology, drug discovery and disease *C Mummery1 1
Leiden University Medical Centre, Department of Anatomy and Embryology, Leiden, The Netherlands
Derivation of heart cells from human pluripotent stem cells (hPSC) is an area of growing interest as a way of modeling disease phenotypes and as a platform for drug discovery and toxicity. Applying the underlying developmental mechanisms that control cardiac differentiation to hPSCs through the use of defined culture conditions in vitro is rapidly moving the field forward: cardiomyocyte differentiation is now a fairly efficient and reproducible process. Genetically marked human embryonic stem cells (hESCs) have been produced in which expression of the green fluorescent protein (GFP) marker is driven by specific lineage markers like Nkx2.5. We are now using these tagged lines in which GFP to select the progenitors of cardiomyocytes, endothelial cells and smooth muscle cells. Applications of hESC- and humaninduced pluripotent stem cell (hiPSC)-derived cardiomyocytes in drug discovery and disease are thus now close to implementation. Results of these studies, in particular drug responses of hPSC-cardiomyocytes to a variety of drugs will be shown. There is an urgent unmet need for reliable cardiac safety pharmacology assays to identify potential risks early in drug development and reduce time and cost to market. The field potential of hPSC-CM can be measured using commercially available multi electrode arrays. Systematic generation of dose response curves for cardiac and non-cardiac drugs show that hPSC-CM accurately predict reported drug effects on the human heart. These include blocking the hERG ion channel, resulting in QT-prolongation; this is associated with life-threatening arrhythmias. Dose responses of a wide range of compounds have been compared and the outcome shown to predict clinical effects. hiPSCs from patients with genetic disease are a source of somatic cells that can carry disease mutations and show disease phenotypes. However, stem cell derivatives are usually immature and whether they represent phenotypes observed in the adult patient and what are appropriate controls, is still a matter of discussion. Data generated using isogenic pairs of hPSC bearing the same cardiac disease mutation as well as a comparison of (mouse) iPSC and ESC-derived cardiomyocytes with adult heart cells will be shown.
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5
Keynote Lecture
KL-006
Reprogramming fibroblasts towards cardiomyocyte-like cells *L Qian1 UNC-Chapel Hill, McAllister Heart Institute, Chapel Hill, NC, USA
1
Cellular reprogramming holds great promise as a novel therapy for heart failure, a common and morbid disease that is usually caused by irreversible loss of functional cardiomyocytes. Because the heart has very limited regenerative potential in response to injury, loss of cardiomyocytes results in impaired pump function and heart failure. Existing treatments are primarily pharmacological and device-based, and do not address the fundamental problem of myocyte loss. Indeed, the prevalence of chronic cardiomyopathy is steadily increasing worldwide, making the identification of novel and effective therapies for this morbid disease an urgent problem in biomedical research. Resident cardiac fibroblasts (CFs) comprise half of the total number of cells in the human heart. The ability to reprogram CFs into functional cardiomyocyte-like cells would theoretically permit regeneration of lost heart tissue. Recently, we showed that introduction of three transcription factors, Gata4, Mef2c and Tbx5 (GMT), directly reprogrammed fibroblasts into induced cardiomyocytes (iCMs) in vivo. In a murine acute myocardial infarction model, delivery of GMT converted CFs into functional iCMs that integrated electrically and mechanically with surrounding myocardium, resulting in a functional improvement and a reduction in scar size. These findings suggest that cellular reprogramming may be an efficient means of regenerating heart tissue in vivo for human patients with heart disease.
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6
Keynote Lecture
KL-007
Quantitative analysis of the cord blood marketplace: size, growth rate, industry drivers, products and worldwide breakout *E Razvi1 1
Select Biosciences LLC, Fremont, CA, USA
This presentation is a quantitative analysis of the cord blood marketplace and provides an analysis of the size, growth rates, as well as industry drivers, and cost-analysis of cord blood banking. SELECTBIO continuing industry coverage of this field focuses upon the market expansion of cord blood utilization based on new protocols and disease classes addressed, as well as the gradual approval of cord blood-based products through the regulatory agencies. SELECTBIO has also characterized the worldwide landscape of cord blood banking and will present a snapshot of these data in this talk.
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7
Keynote Lecture
KL-008
Induction of multi- and pluripotency: innovative approach for neurodegenerative disease modeling and drug screening *HR Schöler1 1
Max Planck Institute for Molecular Biomedicine, Cell and Developmental Biologie, Münster, Germany
Neurodegenerative diseases are severely debilitating, chronic disorders whose prevalence is expected to significantly increase in upcoming years. Despite extensive research, no drugs are currently available for preventing or slowing disease progression, and nearly all therapies under development are suboptimal, with patients remaining symptomatic. Stem cells offer a revolutionary new paradigm for studying neurodegenerative pathogenesis. Using reprogramming, patient-specific induced pluripotent stem cells (iPSCs) can be generated and then differentiated into neurons, recapitulating neurodegenerative pathology in a dish. To better understand the association between mutant LRRK2 and the pathology of Parkinson’s disease (PD), we derived iPSCs from patients with PD harboring LRRK2 G2019S and then specifically corrected the mutant LRRK2 allele. This approach enabled the detection of ERK-dependent dysregulation of novel genes that cause neurodegeneration. However, stem cells also enable a groundbreaking new approach for drug discovery. By automating the disease models, libraries of small molecules can be individually tested for their ability to protect neurons from degeneration. We have recently reported a proof-of-principle study by demonstrating that a stem cell-based assay for neurodegeneration can identify neuroprotective compounds acting through multiple mechanisms. Therefore, taken together, these results demonstrate that stem cells may enable a breakthrough in neurodegenerative research and drug discovery.
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8
Keynote Lecture
KL-009
Normal and neoplastic stem cells *I Weissman1 1
Institute for Stem Cell Biology and Regenerative Medicine, Stanford, CA, USA
Following embryonic development, most of our tissues and organs are continuously regenerated from tissue/organspecific stem cells. The principal property that distinguishes such stem cells from their daughter cells is self-renewal; when stem cells divide they give rise to stem cells (by self-renewal) and progenitors (by differentiation). In most tissues only the primitive stem cells self-renew. Stem cell isolation and transplantation is the basis for regenerative medicine. Self-renewal is dangerous, and therefore strictly regulated. Poorly regulated self-renewal can lead to the genesis of cancer stem cells, the only self-renewing cells in the cancerous tumor. The Weissman lab has followed the progression from hematopoietic stem cells to myelogenous leukemias. They have found that the developing cancer clones progress at the stage of hematopoietic stem cells, until they become fully malignant. At this point, the ‘leukemia’ stem cell moves to a stage of a downstream oligolineage or multilineage progenitor that has evaded programmed cell death and programmed cell removal, while acquiring or keeping self-renewal. While there are many ways to defeat programmed cell death and senescence, there appears to be one dominant method to avoid programmed cell removal – the expression of the cell surface ‘don’t eat me’ protein CD47, the ligand for macrophage SIRP-a. All cancers tested express CD47 to overcome expression of ‘eat me’ signals such as calreticulin and asialogylycoproteins. Antibodies that block the CD47–SIRP-a interaction enable phagocytosis and killing of the tumor cells in vitro and in vivo.
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9
Keynote Lecture
KL-010
Direct lineage reprogramming towards the neural lineage *M Wernig1 1
Stanford School of Medicine, Institute for Stem Cell Biology and Regenerative Medicine, Stanford, CA, USA
Cellular differentiation and lineage commitment are considered robust and irreversible processes during development. Challenging this view, we found that expression of only three neural lineage-specific transcription factors could directly convert fibroblasts into functional in vitro. These induced neuronal (iN) cells expressed multiple neuron-specific proteins, generated action potentials and formed functional synapses. Thus, iN cells are bona fide functional neurons. The question arose whether a defined non-ectodermal cell can be converted into iN cells given the heterogeneity of fibroblast cultures. We therefore tested whether (endoderm-derived) hepatocytes can be reprogrammed to iN cells. Surprisingly, using the same three transcription factors primary mouse hepatocytes could be converted very efficiently into fully functional iN cells. Moreover, gene expression studies on the global and single cell level confirmed not only the induction of a neuronal transcriptional program but also the efficient silencing of the hepatocyte-specific expression pattern. We therefore concluded that iN cells are not hybrid cells with equal identities of the starting cell and neurons, but cells with a predominant neuronal identity with an epigenetic or transcriptional memory of the starting cell population. Completely unexpectedly, the iN cell reprogramming process is substantially more efficient and faster than reprogramming to pluripotent stem cells. We assume that this may be explained by different properties of the transcription factors used for reprogramming. Indeed we found that Ascl1, one of the key driving iN cell transcription factors, is not only a pioneering factor in the sense that it can bind nucleosomal DNA, but it also binds its physiological targets 2 days after infection in mouse fibroblasts. Intriguingly, this property is different from Oct4, Sox2, Klf4, which have been shown to bind nucleosomal DNA but mostly bind ectopic genomic sites. This may be a molecular explanation for the relatively high iN cell conversion efficiencies from mouse fibroblasts.
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10
Keynote Lecture
KL-011
Cell therapy manufacturing process design *D Williams1 1
Loughborough University, Centre for Biological Engineering, Loughborough, UK
This presentation will indicate the range of production technologies available for the manufacturing of cell therapies. It will discuss the how the choice of product technology and business model impacts the corresponding manufacturing strategy and highlight the advantages of the automation of key process steps. Working from the product quality and regulatory requirements on manufacturing the presentation will then show how quality engineering tools can be used to generate more effective process designs. The presentation will close with a perspective emphasising the linkages between regulatory compliance and good manufacturing engineering practice.
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11
Oral Presentation
Biomaterials, scaffolds and nanotechnology
OP-001
Silicon nanoparticles as a perspective tool for biomedical applications: synthesis, physical properties, functional modifications and biocompartibility *E Andreeva1, S Korovin2, A Shubenkov1, V Pustovoy2 & L Buravkova1 1 2
Institute of Biomecal Problems RAS, Cell Physiology, Moscow, Russian Federation General Physics Institute, RAS, Center of Laser Technology and Material Science, Moscow, Russian Federation
In recent decades there has been increased interest in silicon in different fields of human activity as it is electrochemically stable and the second most abundant element in the Earth crust. Its nanoscale application is of special attention due to the possibility of using silicon nanoparticles (Si-NPs) in biomedical purposes. We have developed a new method to obtain Si-NPs and synthesize composite Si-based NPs (Si-C, Si-N, Si-B). The laser-induced pyrolysis setup was constructed that allowed change the reaction parameters in wide range and produce composite NPs with size range 2–20 nm. The produced NPs were stable during several months. The spectral, structural and microscopic measurement were done on the NPs’ size, structure and stoichiometry. The Si-NPs with metal (Ag, Au, Pd) coating were obtained using chemical methods under ultrasound activation. NPs application demand an adequate ex vivo model to evaluate cellular compartibility and biosafety of NPs. Assessment of NPs on cellular level is especially important as its can impact directly onto the cells and intracellular structures. We have tested the effects of pure Si-NPs (7 nm) and modified with B (10 nm) and Pd (15 nm) on mononuclear cells from human peripheral blood (PBMCs) using a multiparameter protocol: side scatter (SS) for intracellular accumulation of NPs, AnnexinV-FITC/PI for cell viabitity and necrotic/apoptotic path of cell death, CM-H2DCFDA for ROS formation, MitoTracer Red FM for transmembrane mitochondrial potential and LysoTracer Green for lysosome activity. Cells were incubated 24 h with NPs in concentration range 1–100 ug/ml. We did not observe cytotoxic effects on PBMCs with any of tested Si- and Si-based NPs. PBMCs retained the granularity, demonstrated by cells w/o NPs. Nevertheless, the cellular organelle analysis demonstrated the stress response, associated with increased production of ROS, changes in the activity of the lysosomal compartment and mitochondrial transmembrane potential. Thus, the Si- and Si-based NPs are promising tool for applications in biomedical technologies due to the absence of cytotoxicity. The presented data demonstrate that besides the direct viability evaluation the comprehensive assessment of the cell stress response should be provided. The intensity of this response can depend on various parameters, such as size, structure and NPs concentration. Financial & competing interests disclosure This work was supported in part by RFBR Grant #11–02–12210-ofi-m-2011.
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12
Oral Presentation
Biomaterials, scaffolds and nanotechnology
OP-002
Nano-fibrous and oriented gelatin scaffolds for nerve tissue engineering *M Buyukoz1, E Erdal2 & S Alsoy Altinkaya3 Izmir Institute of Technology, Bioengineering, Izmir, Turkey Dokuz Eylul University, Medical Biology and Genetics, Izmir, Turkey 3 Izmir Institute of Technology, Chemical Engineering, Izmir, Turkey 1
2
Objectives The incorporation of neural stem cells into scaffolds which could be used to regenerate loss of the cells in the nervous system gives their investigation extreme importance for the treatment of neurodegenerative diseases and traumatic brain injuries. Developing 3D biomimetic scaffolds which provide an optimal environment for cell adhesion, proliferation, differentiation and guide new tissue formation have been major goals in tissue engineering. It is beneficial for a scaffolding material to mimic the characteristics of extracellular matrix at the nanometer scale and to induce natural development for tissue regeneration. Peripheral nerve system is composed predominantly of oriented collagen fibers. Collagen has been used as a nanosized scaffold for tissue engineering but its immunogenicity and pathogen transmission has always been a concern. Gelatin is derived from collagen by acidic or basic hydrolysis and its chemical composition is very similar to that of collagen, thus, it is a good candidate to mimic the chemical composition of natural collagen [1] . Methods In this study, a combination of molding and thermally induced phase separation techniques (TIPS) was used. Molding was employed for creating channeled structure guiding cells during formation of new tissue and TIPS technique was chosen for preparing nano-fibrous gelatin scaffolds which mimic the natural extracellular matrix in terms of nano-size. The effect of gelatin concentration on fiber diameter, fiber length, porosity and mechanical properties of the prepared scaffolds were investigated. As a model neural cell, PC12 was used to observe attachment and proliferation of cells into scaffolds. Results The results have shown that the structure of the channel wall, porosity and mechanical properties of the scaffolds can be easily adjusted by changing polymer concentration and dimensions of the channel. Conclusion Attachment and growth of the PC12 cells in the structure have proposed that this matrix has a potential for neural tissue regeneration. Financial & competing interests disclosure This study was supported by The Scientific and Technical Research Council of Turkey (TUBITAK, Grant Number: 112M568). We also would like to thank Izmir Institute of Technology, Biotechnology and Bioengineering Research and Application Center and Material Research Center for providing experimental support. Reference 1
Liu X, Ma PX. Phase separation, pore structure, and properties of nanofibrous gelatin scaffolds. Biomaterials 30, 4094–4103 (2009).
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13
Oral Presentation
Biomaterials, scaffolds and nanotechnology
OP-003
Functionalization of decellularized scaffold for skeletal muscle tissue engineering *A Costa1,2, P Aprile3,2, P Aulino1, D Rossi1,2, B Perniconi4, S Adamo1, D Coletti4 & L Teodori2 Sapienza University of Rome, Anatomical, histological, forensic medicine and orthopedic sciences, Rome, Italy ENEA-Frascati, Diagnostic and Metrology Laboratory (UPATPRAD-DIM), Frascati, Italy 3 Tor Vergata University of Rome, Molecular Biology, Rome, Italy 4 Université Pierre et Marie Curie Paris 6, UR4 – Ageing, Stress, Inflammation, Paris, Italy
1
2
Objectives Skeletal muscle tissue engineering is a novel, promising tool for repair or substitution of defects arising from traumatic or pathological conditions. Our previous results demonstrated the myogenic potential in vivo of an acellular scaffold derived from a murine hind limb muscle. Although the isogenic orthotopical transplantation led to the formation of new myofibers in the acellular scaffold, a severe inflammatory response inhibited myogenesis and led to precocious reabsorption of the scaffold. Conversely, many cytokines and chemokynes produced during inflammation play a fundamental role in the recruitment and activation of myogenic stem cells. Our research was focused on the functionalization of the acellular scaffold before transplantation in order to modulate the intensity of the inflammation and to enhance myogenesis. Materials & methods We decellularized murine tibialis anterior muscle of cadaveric origin by incubation of dissected muscles in detergent solutions of SDS, Tris and Triton X-100. In order to mask the potential antigenic sites the acellular scaffold was functionalized with polyethylene glycol or murine serum. Alternatively, to reduce the host immune response and enhance de novo production of ECM, we pre-seeded the acellular scaffold with C2C12 myoblasts or an extract of primary satellite cells and fibroblasts. To replace homologous muscles, we orthotopically transplanted decellularized scaffolds, by suturing them to the host tendon extremities following tibialis anterior removal. With this approach the constructs were analyzed in regard to histocompatibility, bioactivity and degradability in vivo. Results The different decellularization protocols tested were less efficient than our method. Scaffolds pre-treated with poly ethylene glycol or mice serum gave rise to an inflammatory response similar or more severe, respectively, than the control. C2C12 pre-seeded scaffold led to a lower inflammation outcome and a more extended myogenesis. We are currently assessing whether transplanted C2C12 are directly responsible for myogenesis or indirectly promote it by diminishing inflammation. A wide repopulation of the acellular scaffold by new regenerating myofibers was obtained by pre-seeding with the extract of primary satellite cells and fibroblasts. Conclusion The decellularization method proposed by us appears to be the most efficient and rapid available so far for skeletal muscle tissue decellularization. The pre-treatment with myogenic cells enhances the potential of the acellular scaffold as a myogenic environment and reduduces the inflammatory response, so it may represent a valid tool for skeletal muscle regenerative medicine.
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14
Oral Presentation
Biomaterials, scaffolds and nanotechnology
OP-005
A new family of self-healing metallophilic hydrogels for tissue engineering application *D Dupin1, P Casuso1, A Pérez1, N Diaz1, I Odriozola2, H-J Grande3, G Cabañero4 & I Loinaz1 IK4-CIDETEC, Biomaterials, Donostia-San Sebastián, Spain IK4-CIDETEC, Nanomaterials, Donostia-San Sebastián, Spain 3 IK4-CIDETEC, Donostia-San Sebastián, Spain 4 IK4-CIDETEC, New Materials, Donostia-San Sebastián, Spain 1
2
Objectives In situ forming hydrogel systems have attracted considerable interest as injectable scaffolds for tissue engineering due to their easy applications and minimally invasive injection procedure. Our research group has recently developed a new generation of injectable supramolecular hydrogels based on low molecular-weight Au (I) and Ag (I) thiolates. Here we present a new family of injectable polymeric hydrogel with selfhealing properties and adjustable mechanical properties used as bone replacement and for intervertebral disc repair. Methods & materials Hydrogel preparation: hydrogels were prepared by mixing an aqueous solution of polymer at with an aqueous solution containing an adjusted amount of metal salt. The amount of polymer was also adjusted to obtain hydrogels at 5, 7, 10 and 20 wt%. Rheological studies: dynamic rheology measurements were performed using a TA Instruments AR G2 temperaturecontrolled rheometer with a 20 mm diameter plate geometry with a solvent trap. Storage modulus (G’) and loss modulus (G’’) were measured with increasing angular frequency (w) at 1% deformation. IVD bioreactor: hydrogel were injected into the nucleus pulposus from a cow’s spine and placed into the bioreactor. Various loads at a frequency of 1 Hz were carried out. Discussions Supramolecular polymeric hydrogels were obtained by mixing an aqueous solution of metals salt (AgNO3 or HAuCl4) with an aqueous solution of thiolated polymer at physiological pH. The time required for gel formation could be easily adjusted, which allows applications of this material as injectable scaffold for tissue engineering, e.g. bone or cartilage replacement. Interestingly, the supramolecular polymeric hydrogel exhibits self-healing properties (Figure 1) due to the reversible interchange between disulfide bonds and supramolecular interaction of thiolates metal at physiological conditions. Moreover, the hydrogels mechanical properties could be tuned on demand by varying the amount of metal ions incorporated into the hydrogel. For example, hydrogels with high amount of added gold exhibit a shear-thickening behavior similar to the synovial liquid. Nevertheless, hydrogels with lower gold concentration show more toughness and could be tested for intervertebral disc repair using a home-made bioreactor where an IV from a cow was filled with our hydrogel. Our results showed such hydrogel could withstand intervertebral disc loads and stress. Moreover, microCT imaging showed that sufate barium-dyed hydrogel did not suffer any deformation and recovered its initial shape, thanks to its self-healing behavior. Separately, hydrogel composites containing bone powder exhibit suitable rheological properties for bone replacement with a shear modulus > 10 MPa. After important loads, hydrogel composites exhibit some visual damage which were completely healed after a few minutes due to the presence of the supramolecular cross-linker. The presence of the allogenic powder should favor bone growth and accelerate bones healing process. Conclusion In summary, this new family of injectable polymeric hydrogels offers advantageous properties for a wide range of applications as a tissue-engineering material such as synovial liquid replacement and intervertebral disc repair. The self-healing properties of those hydrogels allow the full recovery of the material mechanical properties, which allow a longer life time of the scaffold.
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15
Oral Presentation
OP-005
Biomaterials, scaffolds and nanotechnology
Hydrogel
Self-healing in a few seconds
Break
Joined pieces
Figure 1.
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16
Oral Presentation
Biomaterials, scaffolds and nanotechnology
OP-006
3D cell culture using Xanofi’s nanofiber slurries and porous nonwoven substrates *S Gangwal1 1
Xanofi, Research & Development, Raleigh, USA
Many products based on nanofiber scaffolds have been developed to address the rapidly-growing 3D cell culture market to provide a more biologically relevant environment. However, electrospun nanofiber substrates may not offer true three-dimensionality due to their narrow pore openings, which do not allow cells to efficiently migrate into to the depth of a mat substrate. Xanofi has recently developed both nanofiber slurries and porous nanofiber substrates for multiple cell lines that better mimick key features of the extracellular matrix that control migration, proliferation and differentiation of cells. Working with University of North Carolina-Chapel Hill, Xanofi has demonstrated significantly improved cell attachment, proliferation, and chondrogenesis of human umbilical cord mesenchymal stem cells using polylactic acid nanofiber slurries. In collaboration with University College London, it has been demonstrated that the slurry and substrate products show improved growth of HEK293 cells compared with growth on other commercially available nanofiber products. Xanofi’s 3D cell culture products will be discussed for applications in research related to tissue engineering, cancer, stem cell, high-throughput cell culture and regenerative medicine. Further, Xanofi’s novel liquid-based technology, termed XanoShear™, to form nanofibers will be compared with existing methods including electrospinning and melt blowing.
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17
Oral Presentation
Biomaterials, scaffolds and nanotechnology
OP-007
Novel branched cell-penetrating peptides for enhanced delivery of siRNA *J Hoyer1, M Schulz-Siegmund1 & I Neundorf2 University of Leipzig, Institute of Pharmacy, Leipzig, Germany University of Cologne, Institute of Biochemistry, Cologne, Germany
1 2
Objectives Cell-penetrating peptides (CPP) have been extensively studied as a promising tool for the intracellular delivery of various kinds of therapeutic cargo molecules that are otherwise not able to cross the lipid bilayer by themselves due to their size, charge or hydrophilicity. Thus, they present a viable alternative to viral delivery vectors and lipid or polymer based transfection agents in the delivery of nucleic acids. We have previously shown that branched CNPs derived from the peptide hormone human calcitonin (hCT) are able to efficiently transport plasmid DNA into various cell lines as well as primary cells in a non-covalent manner by forming electrostatic complexes with their cargo. However, they were found to lack effectiveness when it comes to small interfering RNA (siRNA) due to insufficient complex formation and endosomal release. Hence, our objective was to introduce suitable modifications such as fatty acids and endosomedisrupting peptide sequences derived from viral membrane fusion proteins to achieve the full potency of hCT-derived carrier peptides also in the delivery of siRNA. Materials & methods All peptides were synthesized by automated standard Fmoc/tBu-solid phase peptide synthesis and obtained with >99% purity as confirmed by analytical RP-HPLC and MALDI-MS. Electrostatic complex formation of positively charged peptides with negatively charged siRNA was investigated by means of an electromobility shift assay and photoncorrelation spectroscopy. Cellular uptake of the carboxyfluorescein-labeled peptides and Alexa Fluor-labeled siRNA was qualitatively and quantitatively followed by fluorescence microscopy and flow cytometry. In order to show the biological effect of intracellularly delivered siRNA, a read-out system comprising HEK-293 transiently transfected with an eYFP-neuropeptide Y Y1 receptor (NPY1R) fusion protein was used. Knockdown following transfection with siRNA targeting NPY1R mRNA was quantitated by fluorescence microscopy on the protein level and real-time RT-PCR on the mRNA level. Cytotoxicity was determined by a resazurin-based cell viability assay. Results Introduction of either decanoic acid or an endosome-disruptive peptide (N-E5L) derived from the HA2-domain of influenza virus to the N-terminus of hCT(18–32)-k7 led to drastically enhanced stability and cellular uptake of electrostatic peptide/siRNA complexes without compromising cell viability. Delivery of siRNA was even achieved in adipose tissue-derived stem cells, which represent usually hard to transfect primary cells. The biological effectiveness of siRNA delivery was proven in transiently transfected HEK-293 cells with knockdown rates of NPY1R expression beyond 50% both on the protein and on the mRNA level. The therapeutic potential is underlined by substantial knockdown of NPY1R also in the breast tumor cell line MCF-7, which endogenously expresses the receptor. Most importantly, peptide-mediated transfection was as efficient as lipofection, which represents the gold standard for siRNA transfection, but often comes along with inherent cytotoxicity. Conclusion We could show that introduction of simple modifications to CPPs can tremendously improve their ability to effectively deliver siRNA into cells and enable hCT-derived CPPs to exploit their full therapeutic potential.
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18
Oral Presentation
Biomaterials, scaffolds and nanotechnology
OP-008
Bottom-up strategy to build up functional 3D dermis equivalent in vitro by tuning microscaffold degradation rate *G Imparato1,2, F Urciuolo1,2, C Casale1,2 & P Netti1,2 University of Naples Federico II, Interdipartimental Research Center on Biomaterials, Naples, Italy Istituto Italiano di Tecnologia, Center for Advanced Biomaterials for Healthcare@CRIB, Naples, Italy
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One of the most challenging issues of in vitro tissue engineering is the implementation of strategies to successfully culture large and viable constructs in vitro, mimicking the natural tissue organization. Most of the natural tissues are often a combination of small repeating units assembled over several scales. As a consequence, from an engineering point of view, it was proposed to build tissues by assembling blocks mimicking those units in a bottom-up or modular approach. We proposed a versatile strategy yielding 3D dermis tissue constructs of defined size and geometry by means of the biological sintering of cell seeded microscaffold so-called microtissue precursors (µTPs). In the present study we produce 3D dermis equivalent exploiting µTPs assembling strategy, and investigate the role of microscaffold properties in guiding ECM organization and assembly towards the realization of the 3D final tissue. To this end we tuned the stiffness and degradation rate of the gelatin microscaffold by varying its crosslinking extent and realizing µTP with each kind of microscaffold type. We hypothesized that microscaffold properties strongly affect the collagen deposition in the extracellular space as well as its assembly and composition, resulting in 3D dermal-like tissue having different final properties. Gelatin porous microcarriers (GPM) were prepared according to a modified double emulsion technique (O/W/O) and then chemically crosslinked by means of glyceraldeide at 3%, 4% and 5% w/w of the microbeads in order to obtain microscaffolds characterized by different degradation rate. Building blocks for bottom-up tissue applications, such as µTPs were obtained by dynamic cell seeding of human dermal fibroblasts on the realized GPM by means of a spinner flask bioreactor. Dynamic culture lasts nine days during which µTPs samples were withdrawn for morphological and histological analyses. At end of spinner culture µTPs were transferred to a maturation chamber where under optimized culture conditions are induced to assembling generating a 3D dermal equivalent tissue. Time and space evolution of de novo synthesized collagen within the 3D growing tissue was monitored by means of histological analyses (staining with Picro sirius Red, hematoxylin eosin and masson’s trichrome), second harmonic generation imaging (SHG) and ultra-structure analyses. Results showed that dynamic seeding performed on GPM at each crosslinking extents led to generation of µTP able to sustain the successively formation of 3D dermal equivalents tissues (Figure 1A) . Endogenous ECM was present in the 3D tissues equivalent realized as highlighted by hystology, polarization microscopy with picrosirius red (Figure 1B) and SHG imaging (Figure 1C) . Imaging analysis performed on picro sirius red staining images of 3D tissue equivalents allowed to discern between thck/mature fiber of collagen and thin/immature fiber of collagen. This quantitative analysis was exploited to monitor collagen maturation confirming that ECM’s time evolution, organization and collagen assembly kinetics strongly depended upon the microscaffold’s crosslinking extent. In particular, the increase of the crosslinking extent sped up collagen assembly kinetic obtaining stiffer ECM able to balance both scaffold’s mass loss and cell traction. The results reached in this study demonstrate that µTP precursor assembly approach can be exploited to build-up human dermal tissue equivalent in vitro completely made up by endogenous ECM components. Due to the fundamental role played by microscaffol’s degradation rate in the collagen maturation, it emerges the importance of controlling initial microscaffold properties in order to obtain 3D tissue having desired final features. A
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Figure 1.
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19
Oral Presentation
Biomaterials, scaffolds and nanotechnology
OP-009
Engineering of a miniaturized biologically vascularized scaffold for three dimensional tissue culture *S Kreß1, J Nickel1 & H Walles1 University Hospital Würzburg, Tissue Engineering and Regenerative Medicine, Würzburg, Germany
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Objectives Regenerative medicine focusses on the full restoration of damaged tissue and utilizes modern issue engineering approaches in order to generate 3D bioartificial tissues which enable or support healing processes by replacing the damaged tissue. If the engineered construct exceeds a critical size a functional vascular network is needed to support a sustained function even after implantation. We already succeeded in engineering a 3D biological vascularized scaffold (BioVaSc) in order to generate bioartificial tissue substitutes and organ replacements. In this study we generated a miniaturized biologically fully vascularized scaffold that on the one hand resembles the features of the already established scaffold and on the other hand meets the demands of down scaled applications. The number of autologous cells needed for colonization of the scaffold can be reduced and, regarding medical applications, less invasive implantations of smaller structured tissues are feasible. Materials & methods Segments of the rat’s small intestine with preserved vasculature were resected and decellularized testing different protocols which mainly utilize sodium deoxycholate and DNase in different incubation times and perfusion rates. The quality of the decellularizing process was confirmed by histological stainings (H&E and Feulgen) and DNA quantification (photospectrometrically). The scaffold was also analyzed proteinbiochemically in order to determine the content of collagens, elastin, glycosaminoglycans and proteoglycans composition. The acellular scaffold was tested concerning its biocompatibility and cytotoxicity by colorimetric MTT assays. By re-seeding the residual vascular structures with microvascular endothelial cells (hMVECs) a revascularization of the scaffold could be achieved. Histological (H&E) and immunohistological (CD31 and vWF) stainings confirmed the endothelial properties of the generated tissue. In addition, tissue viability and metabolic activity was verified by life-dead staining and MTT assays, respectively. Results Concerning the decellularization process the best results were obtained by incubating the rat intestinal segment with ddH2O prior to cell lysis using 4% sodium deoxycholate and DNA cleavage with DNase. A gentle perfusion with all detergents could preserve most of the ECM compared with the native matrix. The scaffold was free of cells and DNA fragments. Furthermore, no cytotoxic effects of the scaffold influencing the metabolic activity of re-seeded cells could be determined. Thus, the vascular structures could be regenerated by the inoculation of microvascular endothelial cells which were afterwards perfused with cell medium through the arterial pedicle for several weeks. Conclusion We miniaturized the established BioVaSc using pieces of the rat intestine in order to generate fully vascularized scaffolds. After decellularization the remaining structures could be used as 3D scaffold for further tissue engineering. The repopulated vasculature could be perfused with cell medium ensuring sufficient nutrient supply for all cells seeded into the scaffold. In further studies the scaffold will be inoculated with additional cell types allowing the generation of a variety of bioartificial tissue substitutes whose functionalities will be tested in vitro. In vivo applicability will be analyzed subsequently in order to use these constructs as implants.
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20
Oral Presentation
Biomaterials, scaffolds and nanotechnology
OP-010
Best decellularization protocol for tissue-engineered lungs *E Kuevda1, E Gubareva1, I Gilevich1, A Sotnichenko1, P Jungebluth2, J Haag2, S Krasheninnikov3, T Grigoriev3, S Chvalun3 & P Macchiarini1,2 Kuban State Medical University, Krasnodar, Italy Karolinska Institutet, Stockholm, Italy 3 Kurchatov Institute, Moscow, Russian Federation 1
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Objective Optimize rat lung decellularization methods using mode-controlled ventilation parameters and combination of nonionic and ionic detergents. Materials & methods In total, 15 adult male Lewis rats, weighing 180 ± 16 g, were used after ethical approval. Native lungs were mounted in bioreactors and treated with different protocols: ventilation via the trachea and retrograde perfusion via the aorta; ventilation via the trachea and anterograde perfusion via the pulmonary artery; and separate perfusion via aorta or pulmonary artery without ventilation. Several combinations and different concentrations of sodium deoxycholate and Triton X-100 were used for cadaveric rat lung decellularization. Air ventilation was performed with Inspira advanced safety ventilator, breath rate and volumes depended on the animal’s weight. Decellularization protocols were based on literature data and on previous experience of our group. The efficiency of decellularization was evaluated by cellular removal by histological staining and DNA assay. Histological evaluation of decellularized lung matrix was performed after formalin fixation for 5 h, processing, embedding in paraffin and sectioning at 5 µm. Sections were stained with hematoxylin and eosin, Masson trichrome, Van Gieson staining for elastic fibers and 4,6-diamidino-2-phenylindole (DAPI) for DNA. Specific matrix components were characterized with immunohistochemistry staining for MHC I, II, collagen I, IV, laminin, elastin, fibronectin after deparaffinization, rehydration and processing with special kits. Ultrastructural analysis of decellularized matrix was performed by scanning electronic microscopy (SEM) after fixation in 2.5% glutaraldehyde in 0.1 M cacodylate buffer. For DNA quantification, standard kit was used at spectrophotometer NanoDrop ND-1000. Native and decellularized lungs underwent biomechanical testing for cyclic compression and relaxation. Results Air tracheal ventilation and antegrade pulmonary perfusion-based 23-h lung decellularization protocol with decreased concentration and timing of ionic detergent (1.5 h of 1% sodium deoxycholate) and additional non-ionic detergent (1 h of Triton-X100) showed no remaining nuclei and preserved matrix architecture. Elastic fibers network and extracellular matrix proteins remained after decellularization. On SEM micrographs, cells were absent but basement membranes were intact. Total DNA was decreased from 581.03 ± 18.42 ng/µg in native lung tissue to 24.73 ± 0.82 ng/µg in decellularized tissue (on average 8% residual presence of DNA in decellularized tissue). Biomechanical tests for stress relaxation and cyclic tests showed completely reversible load both for native and decellularized lungs. Conclusion Air ventilation and antegrade pulmonary perfusion-based protocol with modified detergent treatment is best for lung decellularization due to cellular component elimination and matrix proteins, structure and function preservation.
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21
Oral Presentation
Biomaterials, scaffolds and nanotechnology
OP-011
Titanium oxide-coated cobalt–chromium–molybdenum: improving osteogenesis of mesenchymal stem cells in vitro *N Logan1, L Bozec1, S Collins2, A Traynor2 & P Brett1 University College London, London, UK Corin, Cirencester, UK
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Objectives Titanium (Ti) and its alloys are widely accepted as the prime choice of material for orthopedic and dental applications. However, in some clinical applications the mechanical properties of Ti may not be ideal and other metals such as the cobalt alloy cobalt–chromium–molybdenum (CCM) may be more suitable. Whilst these alloys offer the desired mechanical properties they do not exhibit the same osteogenic biocompatibility as Ti. The objective of this study was to investigate if it is possible to replicate the biocompatibility properties of Ti on CCM by coating it with a layer of TiO2, thus increasing the biocompatibility of CCM, whilst maintaining its desirable mechanical properties. Methods Human mesenchymal stromal cells (MSCs) derived from three donors were incubated at standard culture conditions of 37°C, 5% CO2 in MEM with media changes every 3–4 days. Cells were harvested when they reached 80–90% confluence and only used up to passage 6. The CCM discs were initially supplied with a machined surface topography (Corin Ltd, UK), and were ground and polished to a smooth finish with Struers RotoForce-1 machines using a combination SiC 1000# grit paper and 3 µm DP-Suspension P solution on MD-Dac to ensure consistent surface topographies amongst the discs. The discs were subsequently coated with a thin layer of TiO2 (~100nm thick) by chemical vapour deposition (CVD). Smooth Ti discs were used as a positive control surface and smooth CCM discs were used as a negative control surface. Characterisation of the samples included contact angle measurements and surface roughness analysis. All samples underwent UV sterilisation before the cell culture experiments. In order to assess the osteogenic activity of the cells, calcium and collagen deposition assays were performed on CCM, TiO2 coated CCM (CCMT) and Ti discs in triplicate. To investigate the internal structure of the MSCs, confocal microscopy was used to monitor cytoskeletal changes in the cells at 1 and 7 days post seeding, using propidium iodide and phalloidin to fluorescently stain the nuclei and F-actin respectively. Immunohistochemical analysis of hydroxyapatite and collagen deposition was performed at 7, 14 and 21 days. Results On week 2 of the calcium mineralisation assay, CCMT was shown to deposit significantly more calcium than on the CCM (p > PBS). In addition, we confirmed that the ACV membrane using SFM could provide almost equal properties of traditional native CV membrane using CM-FBS. Financial & competing interests disclosure This work was supported by a grant to T Takezawa for Agri-Health Translational Research Project (No. 6110) from the Ministry of Agriculture, Forestry and Fisheries of Japan. Information resources 1
Takezawa T, Ozaki K, Nitani A, Takabayashi C, Shimo-Oka T. Collagen vitrigel: a novel scaffold that can facilitate a three-dimensional culture for reconstructing organoids. Cell Transplant. 13, 463–473 (2004).
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Takezawa T, Takeuchi T, Nitani A et al. Collagen vitrigel membrane useful for paracrine assays in vitro and drug delivery systems in vivo. J. Biotechnol. 131, 76–83 (2007).
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Takezawa T et al. A novel material of high density collagen fibrils: a collagen xerogel membrane and its application to transplantation in vivo and a culture chamber in vitro. In: 24th European Conference on Biomaterials (International Proceedings Division). Medimond, Bologna, Italy, 181–185 (2012).
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26
Oral Presentation
Biomaterials, scaffolds and nanotechnology
OP-015
Layer-by-layer coated microcarriers as drug-delivery-system: a new approach for treatment of chronic inflammations *U Reibetanz1, S Rathmann1, M Schönberg1, V Strehlow1, M Göse1 & J Lessig2 University of Leipzig, Medical Faculty, Institute for Medical Physics and Biophysics, Leipzig, Germany University Hospital Giessen and Marburg, Institute for Pathology, Giessen, Germany
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Objectives Chronic inflammations are characterized by a massive invasion of polymorphonuclear leukocytes (PMNs) into inflammatory loci resulting in an uncontrolled release of highly reactive substances. This invasion complicates the elimination of apoptotic PMNs and subsequently the termination of the inflammatory process [1] . Current anti-inflammatory systemic approaches are often accompanied by unwanted side effects. Therefore, layer-by-layer (LbL) biopolymer coated colloidal microcarriers represent a new approach for targeted delivery of highly specific anti-inflammatory substances. Due to the modular design, the carriers can be equipped with a defined amount of active agents and further functionalized to provide a targeted transport to the desired cells and a time-controlled release of the agents. First investigations are focused on the application of the protease inhibitor a1-antitrypsin (AT) in order to reduce the damaging effects of highly reactive elastase (HNE). The emphasis here is on the simultaneous extra and intracellular application of AT for enhanced inhibitory efficiency. Materials & methods CaCO3 particles were used as templates and LbL coated with protamine sulphate and dextran sulphate. Controlled dissolution of the templates for capsule production was monitored using a fluorescence tool to observe [2] remaining Ca 2+. AT was assembled onto the surface as well as integrated within different layer depths and monitored by flow cytometry and confocal laser scanning microscopy via fluorescence labels. HNE activity after AT-carrier application was then photometrically detected. Results & discussion By applying functionalized microcarriers to PMNs, it has to be ensured that intracellular active carriers undergo a fast uptake in order to release the agents during the short PMN’s life time and also do not contribute to proinflammatory signalling in macrophages. A fast interaction and uptake within 2–4 h as well as a time-dependent multilayer decomposition up to five layers under PMN’s phagolysosomal/granular conditions2 reveal the delivery potential of the carriers within the cells. Investigations of carrier (particles and capsules) interaction with macrophage-like cells U937 show, that low cell/carrier ratios result in only marginal proinflammatory response (low TNF-a, IL-1b release) [3,4] . Since HNE is both stored in PMN granules and permanently released from the cells, an optimal inhibitory efficiency of AT towards HNE can be expected by a simultaneous application of AT-functionalized carriers within phagolysosomes of PMNs and the extracellular space. Assembling a defined amount of AT as outermost layer, the coincubation of the carriers with PMNs shows a strong inhibitory effect towards HNE. Compared with carrier/supernatant interactions, it could be shown that HNE inhibition is attributed to both, extra- and intra-cellular AT efficacy [5] . In conclusion, AT loaded microcarriers can be efficiently applied to PMNs for a dual inhibition of HNE in order to prevent the damaging effects of PMNs in chronic inflamed tissue and regenerate tissue homeostasis. References 1
Haslett C. Granulocyte apoptosis and inflammatory disease. Br. Med. Bull. 53, 669–683 (1997).
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Rathmann S, Schönberg M, Lessig J, Reibetanz U. Interaction, uptake, and processing of LbL-coated microcarriers by PMNs. Cytometry A 79(12), 979–989 (2011).
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Lessig J, Neu B, Reibetanz U. Influence of layer-by-layer (LbL) assembled CaCO(3)-carriers on macrophage signaling cascades. Biomacromolecules 12, 105–115 (2011).
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Strehlow V, Lessig J, Göse M, Reibetanz UJ. Mater. Chem. B doi:10.1039/C3TB20390E (2013) (Epub ahead of print).
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Reibetanz U, Schönberg M, Rathmann S, Strehlow V, Göse M, Leßig J. Inhibition of human neutrophil elastase by a1-antitrypsin functionalized colloidal microcarriers. ACS Nano 6, 6325–6336 (2012).
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27
Oral Presentation
Biomaterials, scaffolds and nanotechnology
OP-016
Functionalized electrospun nanofibers for the development of basement membranes *A Rossi1, H Walles1 & J Groll2 University Hospital Würzburg, Department of Tissue Engineering and Regenerative Medicine, Wuerzburg, Germany 2 University Hospital Wuerzburg, Department of Functional Materials in Medicine and Dentistry, Wuerzburg, Germany 1
Objective Basement membranes (BM) are layered, 50–100-nm-thick, cell-adherent extracellular matrices. Initially believed to serve as a selective barrier to which cells adhere, it has become evident that the individual components of the BM are regulators of biological activity such as cell growth, differentiation and migration. Laminin and collagen IV are the major constituents of BM. Cells have specific receptors for these matrix components. A pentapeptide within the cell binding domain of laminin has been found to adhere to a specific cell surface receptor and in this way mediate cell adhesion. Collagen IV also contains a cell attachment domain, which is involved in its binding to the receptor. Electrospinning is well known as an efficient technique for the fabrication of polymer nanofibers and therefore a suitable method to produce an artificial BM. One-step addition of a functional, amphiphilic macromolecule as star-shaped poly(ethylene oxide) derivate transforms electrospun hydrophobic PLGA fibers into hydrophilic fibers and makes the attachment of these cell-adhesion mediating peptides possible. As a result, this will enable the formation of biomimetic in vitro BM, which plays an important role in many physiological processes. Materials & methods Electrospinning was performed with a solution of 6 w/v% NCO-sP(EO-stat-PO) and 28 w/v% PLGA RG 504 in a mixture of acetone, DMSO and acidic water. The solution was spun at a feed rate of 0.5ml/h through a flat-tip stainless steel spinneret connected to a high-voltage power supply. Fibrous mats were collected on a rotating drum at a distance of 160 mm. Fibers were modified with binding motifs of collagen IV and laminin, which are the two major components of the BM. Results Covalent attachment of GRGDS, a cell-binding domain of fibronectin to hydrophilic PLGA fibers promotes specific bioactivation and enables adhesion of fibroblasts through exclusive recognition of the immobilized binding motif, whereas the negative control peptide GRGES prevents cell adhesion. In cell culture experiments it was shown that fibers can also be modified with laminin and collagen IV sequences. Coculture experiments with HaCaT cells on the one side and fibroblasts on the other side have validated that it is possible to create skin equivalents with these functionalized fibers. The keratinocyte line grows in several layers and expresses cytokeratin 10 in the stratified epithelium; cytokeratin 14 was secreted in direction to the BM. A critical function of BM is to mediate cell adhesions to different cell types through polarization of the cells. By expressing cytokeratin 14, a marker for cells still proliferating, in the basal layers and cytokeratin 10 in the upper layers, it was shown that polarization, which contributes to cell organization and stability within developing tissues, is existing in our artificial BM. Vimentin staining of the fibroblasts showed that the cells infiltrated into the electrospun membrane. Conclusion Initially believed to serve as a selective barrier and scaffold to which cells adhere, it has become evident that the individual components of the BM are regulators of biological activities such as cell growth, differentiation and migration and that they influence tissue development and repair. The establishment of a method to make specific cell adhesion on electrospun fibers possible offers ample opportunity for the construction of bipolar membranes. These membranes are suitable for both in vitro coculture systems and complex artificial BM.
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28
Oral Presentation
Biomaterials, scaffolds and nanotechnology
OP-017
Skin and bone integrated pylon (SBIP) seeded with autologous fibroblasts for direct skeletal attachment of limb prostheses *M Shevtsov1,2, N Yudintceva2, M Blinova2, O Galibin1, A Ivanova3, O Savchenko3, G Pinaev2, I Potokin4 & M Pitkin5,6 IP Pavlov State Medical University, Biotechnology Department, St Petersburg, Russian Federation Institute of Cytology of the Russian Academy of Sciences, Department of Cell Technology, St Petersburg, Russian Federation 3 Russian Research Center of Radiology and Surgical Technology, Department of radiology, St Petersburg, Russian Federation 4 Research Institute of Highly Pure Biopreparations, Department of Microscopy, St Petersburg, Russian Federation 5 Tufts University, Boston, MA, USA 6 Poly-Orth International, Sharon, MA, USA 1
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Objectives Periprosthetic infectious complications due to percutaneous implantation constitute the major problem in direct skeletal attachment of limb prostheses. A two-component porous composite skin and bone integrated pylon (SBIP) intended for a two-step (intraosseous and transdermal) implantation was developed. To improve the skin-implant and the boneimplant bond we applied fibroblasts. The biocompatibility and feasibility of the two-stage implantation of SBIP seeded with autologous fibroblasts was evaluated in animal study. Materials & methods The first study was to investigate an intramedullar implantation only. The purpose was to assess the effectiveness of the fibroblast treatment of the intramedullar component of the SBIP-TS kit on the implant–bone interface. In addition, we wanted to learn if an induction of the fibroblasts into osteoblasts differentiation would be more beneficial for further osseointegration. After above-knee amputation in rabbits, we implanted intramedullary component treated or not with fibroblasts. The processes of angio- and osteo-genesis were assessed with the help of three-phase scintigraphy at 1, 2, 4 and 8 weeks after the implantation with subsequent histological assay. The follow-up period constituted 9 months. In the second part of the study we evaluated the role of fibroblasts on the biointegration of the transcutaneous component attached to the first one at the level of the derma. After 6–8 weeks from the implantation of the intramedullary component, we attached the transcutaneous part of the prosthesis pretreated with fibroblasts in collagen gel. After 2 months several elected animals were sacrificed and assessed histologically. The follow-up period constituted 5 months. Results We observed the increased osseointegrative properties of the intramedullary titanium porous component seeded with the fibroblasts induced into the osteoblasts differentiation compared with the untreated porous titanium pylon. The three-phase scintigraphy and subsequent histological analysis showed that the level of osteogenesis was 1.5-fold higher then in the control group (p
