Osteoarthritis (OA), a prevalent chronic condition with a striking impact on life quality,
represents an enormous societal burden that increases greatly as populations’ age. Yet
no approved pharmacological intervention, biologic therapy or procedure prevents the
progressive destruction of the OA joint. Based on bilayered structures that have been
previously suggested for osteochondral (OC) applications (Oliveira et al 2006) and on
the potential of methacrylated gelatin (GelMA) and methacrylated gellan gum (MAGG)
for different tissue engineering applications (Silva-Correia et al 2013, Tasoglu et al
2014), we set a dynamic platform for the in vitro recreation of an OA 3D in vitro model.
Since OA is an inflammatory and degenerative disorder affecting cartilage and
subchondral bone, we created 6 hybrid formulations recreating a 3D controlled
subchondral bone and cartilage integrated microenvironment. Fat pad adipose derived
stem cells (ASCs) were isolated from Hoffa’s body obtained from healthy Patients,
characterized by flow cytometry and their performance in the developed 3D structures
assessed. GelMA formulation showed the best cell adhesion and proliferation, but the
life-time of this one in culture is shorter due to the faster degradation in vitro comparing
to MAGG based structures. According to this we proceeded with the best hybrid
formulation, GelMA-MAGG 2:1, for OC co-differentiation using a dual-chamber
bioreactor designed for the establishment of co-cultures in a single 3D structure
(Canadas et al 2014). This approach solved challenges of 3D cell culture in interfaced
tissues as OC and will ultimately be used for OA in vitro modelinginfo:eu-repo/semantics/publishedVersio

Bullpit, AJ

Carey, D

Finlay, S

Kirkham, J

Seedhom, BB

Treanor, DE

Canadas, Raphael Faustino

Marques, A. P.

Oliveira, J. M.

Reis, R. L.

Demirci, U.

Universidade do Minho: RepositoriUM

Dynamic Platform To Recreate An Osteoarthritic 3D In Vitro Model RF Canadas1, 2, AP Marques1, JM Oliveira1, RL Reis1, U Demirci2 1 3B’s Research Group – Biomaterials, Biodegradable and Biomimetics, Headquarters of the European Institute of Excellence on tissue Engineering and Regenerative Medicine, AvePark, 4805-17 Barco, Guimarães, Portugal; ICVS/3B’s- PT Government Associate Laboratory, University of Minho, Guimarães- Braga, Portugal 2 Bio-Acoustic MEMS in Medicine (BAMM) Laboratory, Canary Center at Stanford for Cancer Early Detection, Department of Radiology, Stanford School of Medicine, Palo Alto, California 94304, USA 2016/12/1 TISSUE ENGINEERING PART A Vol. 22 S67-S67 MARY ANN LIEBERT, INC  Osteoarthritis (OA), a prevalent chronic condition with a striking impact on life quality, represents an enormous societal burden that increases greatly as populations’ age. Yet no approved pharmacological intervention, biologic therapy or procedure prevents the progressive destruction of the OA joint. Based on bilayered structures that have been previously suggested for osteochondral (OC) applications (Oliveira et al 2006) and on the potential of  methacrylated gelatin (GelMA) and methacrylated gellan gum (MAGG) for different tissue engineering applications (Silva-Correia et al 2013, Tasoglu et al 2014), we set a dynamic platform for the in vitro recreation of an OA 3D in vitro model. Since OA is an inflammatory and degenerative disorder affecting cartilage and subchondral bone, we created 6 hybrid formulations recreating a 3D controlled subchondral bone and cartilage integrated microenvironment. Fat pad adipose derived stem cells (ASCs) were isolated from Hoffa’s body obtained from healthy Patients, characterized by flow cytometry and their performance in the developed 3D structures assessed. GelMA formulation showed the best cell adhesion and proliferation, but the life-time of this one in culture is shorter due to the faster degradation in vitro comparing to MAGG based structures. According to this we proceeded with the best hybrid formulation, GelMA-MAGG 2:1, for OC co-differentiation using a dual-chamber bioreactor designed for the establishment of co-cultures in a single 3D structure (Canadas et al 2014). This approach solved challenges of 3D cell culture in interfaced tissues as OC and will ultimately be used for OA in vitro modeling. 

Dynamic platform to recreate an osteoarthritic 3D in vitro model

More than 200,000 peripheral nerve repair procedures are performed
annually in the USA. But, the adequate functional recovery of
the peripheral nerve is still a significant clinical challenge. The direct
suture repair without the use of grafted materials may be used in cases
where a short (<5 mm) nerve gap has to be overcome. However, larger
defects repaired by neurorrhaphy, exhibit excessive tension over the
suture line and offer poor surgical results. The nerve autograft is
recognized as the ‘‘gold standard’’ technique but it is limited by tissue
availability, donor-site morbidity, secondary deformities, as well as
potential differences in tissue structure and size. In our group, we have
been developing a series of natural-based biomaterials to be used as
nerve conduits that became an alternative to synthetic polymers such
as PCL, PLGA and polyurethane. Among the natural materials,
chitosan, Gellan gum, keratin and silk fibroin have been used. The
relevant in vitro and in vivo studies that have been performed will be
presented. Their final properties (e.g., compressive modulus, storage
modulus, stiffness, swelling behavior, durability, degradation profile,
porosity, permeability, suture ability) can be tuned for specific uses,
by means of using different concentrations and processing techniques,
i.e. fibres, membranes and tubes can be produced. Furthermore, the
inner diameter, thickness of the wall and length of the nerve guidance
conduit can be tuned according to the final needs (e.g. permeability
and biodegradability), thus opening up new possibilities to address the
current challenges in PNR, especially in the treatment of long gapsinfo:eu-repo/semantics/publishedVersio

Carvalho, C. R.

Costa, J. B.

follow. In this study, gas foaming method in a double emulsion-solvent were used to fabricate the well interconnected porous PLGAmicrosheres. Several factors were adjusted to acquire proper celldelivery construct. In order to promote cell adhesion, we use deso-lvation to coat silk protein onto the surfaces of the as-prepared mi-crospheres. Gingival stem cells were seeded on the silk modifiedPLGA microspheres to evaluate cell viability and osteogenic po-tentials. Visualization and biochemical analyses of the microcarrier-cell constructs were performed to demonstrate cell proliferation andphenotypic expression. EdU analysis displayed better proliferation ofthe stem cells on the silk modified microspheres. The calcium nod-ules were observed on the microspheres after 28 days’ osteogenicinduction. Deposition of the calcium on the spheres was proved byalizarin staining. EDS analysis demonstrated that calcium andphosphate was deposited on the microspheres, and the XRD resultsproved that the calcium nodules were formed by hydroxyapatitecrystals. In conclusion, injectable silk modified PLGA microspheresare promising cell delivery constructs and for other applications,especially for bone regeneration.405Polyelectrolyte Anionic Complexes Of Chitosan-alginate-aloeVera: Characterization And Determination Of AntimicrobialActivity And Lymphocyte Culture ViabilityK. Shirai1, R. Gallardo1, C. Pérez-Alonso2, H. Vázquez-Torres3,A. Roman1, M. Aguilar-Santamarı́a1, E. Vázquez1;1Biotechnology, Universidad Autónoma Metropolitana, MexicoCity, MEXICO, 2Chemical engineering-Campus Cerrillo,Universidad Autónoma del Estado de México, Toluca, MEXICO,3Physical, Universidad Autónoma Metropolitana, Mexico City,MEXICO.Polyelectrolyte complexes (PECs) of Aloe vera (Aloe barbadensisMiller) (AV) and alginate (ALG) were formulated at pH 6 with andwithout Ca ions using commercial chitosan (QCH) prepared byconventional thermochemical methods and chitosan obtained frombiologically extracted chitin (BCH). The blends presented poly-anionic behavior with the zeta potential (z) determination allowing astable system, this is important for biological applications due tointeractions with cell membranes are minimized and avoiding per-meation disturbance. Cell viability was evaluated using lymphocytesculture through MTT and RN assays, which were up 80% in bothtests. Fluorescence microscopy analysis evidenced the formation ofmicrogels in the systems due to the fluorescence produced by theinteraction of CH from PECs with calcofluor white reagent. Scanningelectron microscopy analysis showed disordered morphology withrough and lamellar structures. The PECs-BCH with Ca+2 ions showeda relatively smooth porous structure, whereas PECs-QCH with Ca+2had a macro rough porous network with pore sizes between 10 and20mm. Moreover, the structure of the PEC was strongly influenced bythe amount of Ca+2 ions, thus leading to a more compact, homoge-neous and less dense structure. This difference between PECs at pH 6with and without Ca2+ ions might be due to degree of acetylation (DA)of CH, since PECs-QCH-6-Ca presented higher DA than PECs-BCH-6-Ca. PECs showed antimicrobial activity on Staphylococcus epi-dermidis as it was determined in the live/dead fluorescent assay.406Human Derived Cells And Biomaterials For Tissue EngineeringAnd RegenerationH. Redl1,2,3, W. Holnthoner1,2,3, V. Hruschka1,2,3,A. Lindenmair1,2,3, R. Mittermayr1,2,3, E. Priglinger1,2,3,P. Slezak1,2,3, S. Wolbank1,2,3;1Ludwig Boltzmann Institute for experimental and clinicalTraumatology, Vienna, AUSTRIA, 2AUVA Research Center,Vienna, AUSTRIA, 3Austrian Cluster for Tissue Regeneration,Vienna, AUSTRIA.Human plasma derived fibrin matrix is one of the most versatilebiomaterials for tissue engineering and regenerative medicine.Working for over 30 years in this field we have gained notable ex-perience and we can demonstrate the advantages and limitations,application techniques and the special use for growth factor and celldelivery as well as a gene activated matrix. Another aim of our groupis to use, medical garbage’’ for regenerative purposes. Therefore, weuse cells from liposuction, from umbilical cord and placenta derived(PD) - substances (e.g. collagen) and PD structures (e.g. amnion) aswell as PD cells (e.g. amnion MSC). ‘‘Living’’ amnion is used eitherdirectly (cryopreserved) by using a clinically approved process, e.g.for wound healing and antifibrosis, or in a new process where thestem cells residing on and in amnion (‘‘sessile’’ cells) are differen-tiated in toto (osteo, chondrogenic direction). Isolated stem cells arecultured with platelet derived factors (from outdated platelets) toavoid animal products and used directly or pre-differentiated, inautologous or allogeneic fashion. Allogeneic is possible, because themesenchymal stem cells have minor antigenicity and, in addition,immunosuppressive properties. This talk aims to provide an over-view of the use of the above mentioned procedures within the Aus-trian Cluster for Tissue Regeneration.407Advanced Natural-based Biomaterials To Tackle The CurrentChallenges In Peripheral Nerve RegenerationJ. M. Oliveira, Sr.1, C. R. Carvalho1, J. B. Costa2, R. L. Reis, Sr.2;1UMinho, Braga, PORTUGAL, 23Bs, UMinho, Braga,PORTUGAL.More than 200,000 peripheral nerve repair procedures are per-formed annually in the USA. But, the adequate functional recovery ofthe peripheral nerve is still a significant clinical challenge. The directsuture repair without the use of grafted materials may be used in caseswhere a short (<5 mm) nerve gap has to be overcome. However, largerdefects repaired by neurorrhaphy, exhibit excessive tension over thesuture line and offer poor surgical results. The nerve autograft isrecognized as the ‘‘gold standard’’ technique but it is limited by tissueavailability, donor-site morbidity, secondary deformities, as well aspotential differences in tissue structure and size. In our group, we havebeen developing a series of natural-based biomaterials to be used asnerve conduits that became an alternative to synthetic polymers suchas PCL, PLGA and polyurethane. Among the natural materials,chitosan, Gellan gum, keratin and silk fibroin have been used. Therelevant in vitro and in vivo studies that have been performed will bepresented. Their final properties (e.g., compressive modulus, storagemodulus, stiffness, swelling behavior, durability, degradation profile,porosity, permeability, suture ability) can be tuned for specific uses,by means of using different concentrations and processing techniques,i.e. fibres, membranes and tubes can be produced. Furthermore, theinner diameter, thickness of the wall and length of the nerve guidanceconduit can be tuned according to the final needs (e.g. permeabilityand biodegradability), thus opening up new possibilities to address thecurrent challenges in PNR, especially in the treatment of long gaps.408The Effects of Nicotine Oils, Collagen and Fibronectinon the Initial Adhesion, Cell Proliferation and MorphologyOf Human Mesenchymal Stem CellsB. M. Hull, A. Sassen, M. Walcott, R. Anand;Extended University, California State University Channel Islands,Camarillo, CA.Introduction: Integrins are the main receptors on cell membranesthat bind to the extracellular matrix (ECM) stimulating cell growthand proliferation. Fibronectin is a glycoprotein which is part of theextracellular matrix that bind to integrins. Fibronectin promotescellular adhesion, fusion, and migration in vitro for several models.Collagen is the main structural protein in extracellular space invarious connective tissues in most tissues. A fairly new product onS-106 2016 AM ABSTRACTS

Tissue Engineering Part A

English

Advanced natural-based biomaterials to tackle the current challenges in peripheral nerve regeneration

Holmes, RA

Yang, X

Tronci, G

Wood, D

White Rose Research Online

This is a repository copy of Click-crosslinkable Collagen Hydrogels For Cytocompatible 3DCulture In Regenerative Medicine.White Rose Research Online URL for this paper:http://eprints.whiterose.ac.uk/111921/Version: Accepted VersionProceedings Paper:Holmes, RA, Yang, X orcid.org/0000-0002-0144-2826, Tronci, G orcid.org/0000-0002-9426-4220 et al. (1 more author) (2016) Click-crosslinkable Collagen Hydrogels For Cytocompatible 3D Culture In Regenerative Medicine. In: Tissue Engineering Part A. TERMIS - Americas Conference & Exhibition, 11-14 Dec 2016, San Diego, USA. Mary Ann Liebert , S126-S126. https://doi.org/10.1089/ten.tea.2016.5000.abstracts(c) Mary Ann Liebert, Inc. This is an author produced version of an abstract published in Tissue Engineering Part A. Uploaded in accordance with the publisher's self-archiving policy. Final publication is available from Mary Ann Liebert, Inc., publishers at: https://doi.org/10.1089/ten.tea.2016.5000.abstractseprints@whiterose.ac.ukhttps://eprints.whiterose.ac.uk/Reuse Unless indicated otherwise, fulltext items are protected by copyright with all rights reserved. The copyright exception in section 29 of the Copyright, Designs and Patents Act 1988 allows the making of a single copy solely for the purpose of non-commercial research or private study within the limits of fair dealing. The publisher or other rights-holder may allow further reproduction and re-use of this version - refer to the White Rose Research Online record for this item. Where records identify the publisher as the copyright holder, users can verify any specific terms of use on the publisher’s website. Takedown If you consider content in White Rose Research Online to be in breach of UK law, please notify us by emailing eprints@whiterose.ac.uk including the URL of the record and the reason for the withdrawal request. 483 Click-crosslinkable Collagen Hydrogels For Cytocompatible 3D Culture In RegenerativeMedicineR. A. Holmes, X. Yang, G. Tronci, D. Wood;Department of Oral Biology, University of Leeds, Leeds, UNITED KINGDOM.Hydrogels provide a three-dimensional (3D) framework with tissue-like elasticity for culturing cells inregenerative medicine. Injectable materials delivered in aqueous solution are considered ideal for theintroduction of cells and bioactive factors through minimally invasive methods to fill complex 3D shapes.A novel injectable click-crosslinkable collagen-based hydrogel has been created capable of cellencapsulation in vitro/in vivo. The objectives were to prepare a novel cross-linking strategy for theformation of hydrogels with varied mechanical and physical properties to examine differentiation andproliferation with dental pulp stem cells (DPSCs). Collagen was thiolated by a substitution reaction usingTraut's reagent at pH 7.4 without the need of an additional base. The degree of functionalization wasquantified using a TNBS assay and circular dichroism was used to confirm the ratio of positive tonegative peaks (RPN) was comparable to native collagen. The hydrogels were formed in 10 minutes by athiol-ene photo-click reaction using violet light (405ௗnm) between the thiolated collagen andpoly(ethylene glycol) norbornene. Varied mechanical and physical properties of the gels have been madeby varying the molar ratio of SH: norbornene and the degree of thiol functionalization. The effect ofdegradation on the mechanical and physical properties were studied using both simulated body fluid andcollagenase. The prepolymer solution was prepared in PBS and DPSCs and G292 cells were encapsulatedin the gel in vitro. Cell spreading, cell migration and cell survival were analysed using ATP assay andLIVE/DEAD staining.http://online.liebertpub.com/doi/full/10.1089/ten.tea.2016.5000.abstracts?mobileUi=02016 TERMIS – Americas. Conference and Exhibition, San Diego, CA. December 11–14,2016To cite this article: Tissue Engineering Part A. December 2016, 22(S1): S-1-S-156.doi:10.1089/ten.tea.2016.5000.abstracts.Published in Volume: 22 Issue S1: December 1, 2016

Click-crosslinkable Collagen Hydrogels For Cytocompatible 3D Culture In Regenerative Medicine

This is a repository copy of The Benefits of Objective, Quantitative Image Analysis of Histological Sections in Tissue Engineering Studies.White Rose Research Online URL for this paper:http://eprints.whiterose.ac.uk/111945/Version: Accepted VersionProceedings Paper:Finlay, S, Carey, D, Bullpit, AJ et al. (3 more authors) (2016) The Benefits of Objective, Quantitative Image Analysis of Histological Sections in Tissue Engineering Studies. In: Tissue Engineering Part A. TERMIS - Americas Conference & Exhibition, 11-14 Dec 2016, San Diego, USA. Mary Ann Liebert , S73-S73. https://doi.org/10.1089/ten.tea.2016.5000.abstracts(c) Mary Ann Liebert, Inc. This is an author produced version of an abstract published in Tissue Engineering Part A. Uploaded in accordance with the publisher's self-archiving policy. Final publication is available from Mary Ann Liebert, Inc., publishers at: https://doi.org/10.1089/ten.tea.2016.5000.abstractseprints@whiterose.ac.ukhttps://eprints.whiterose.ac.uk/Reuse Unless indicated otherwise, fulltext items are protected by copyright with all rights reserved. The copyright exception in section 29 of the Copyright, Designs and Patents Act 1988 allows the making of a single copy solely for the purpose of non-commercial research or private study within the limits of fair dealing. The publisher or other rights-holder may allow further reproduction and re-use of this version - refer to the White Rose Research Online record for this item. Where records identify the publisher as the copyright holder, users can verify any specific terms of use on the publisher’s website. Takedown If you consider content in White Rose Research Online to be in breach of UK law, please notify us by emailing eprints@whiterose.ac.uk including the URL of the record and the reason for the withdrawal request. The Benefits of Objective, Quantitative Image Analysis of Histological Sectionsin Tissue Engineering StudiesScott, Finlay (University of Leeds)Bahaa, B Seedhom (University of Leeds)Duane, O Carey (University of Leeds)Andy, J Bulpitt (University of Leeds)Darren, E Treanor (University of Leeds and NHS)Jennifer, Kirkham (University of Leeds)Introduction: Recently we showed the potential of a self-regulating mechanicalloading regime to form constructs with cartilage-like histology and high stiffness1.Histology usage in tissue engineering is predominately qualitative, restricting utilityand data exploitation/interpretation. Our aim was to determine any correlation ofmetrics derived from objective and quantitative image analysis with functionalcharacteristics of cartilage constructs.Materials and Methods: Sections of tissue engineered cartilage1 were stained witheither Alcian blue/Sirius red, Harris’s haematoxylin or antibodies for type I or type IIcollagen, and scanned to produce ultra-high-definition images. Percentage coverageand mean and median staining intensity were determined using custom software,and compared with compressive moduli. In addition to this, a novel method of nucleidetection and degree of eccentricity (circular vs elliptical) was developed.Results: Positive correlations with moduli were found for type II collagen staining(R2:0.56-0.59) and Alcian blue staining (R2:0.85-0.87), mirroring previousbiochemical data. Nuclei became more circular as constructs became moredeveloped; inferring a chondrocyte phenotype.Discussion and Conclusions: Quantitative image analysis provides added value toresource intensive, routine histology and correlates with functional properties oftissue engineered constructs, increasing data utility and aiding interpretation.Reference: Finlay et al., In Vitro Engineering of High Modulus Cartilage-LikeConstructs, Tissue Engineering: Part C, 22:4, 2016.

The Benefits of Objective, Quantitative Image Analysis of Histological Sections in Tissue Engineering Studies

Therapeutic deep eutectic systems (THEDES) are here proposed as a new class of pharmaceutical active ingredient (API) whose distinctive characteristic is the fact that they are liquid at room temperature. Eutectic systems have been described in the pharmaceutical sciences as an alternative formulation able to enhance bioavailability of the API. THEDES are constituted by one active agent and a coformer or two active agents, which when combined at a particular molar ratio become liquid at room temperature. In this work, we explore the prepararation of the THEDES menthol-ibuprofen. The solubility and dissolution profile in phosphate buffer solution (pH 7.4) was performed at 37Â°C and compared with those of the pure API. Furthermore, the in vitro permeability of the liquid THEDES and the API in powder form, was evaluated using a Franz diffusion cell and caco-2 cells as model of human intestinal epithelium. The solubility of the API's when in the THEDES system improved as much as up to 12 fold. Furthermore, for this system the permeability was calculated to be 14â  Ã â  10â  5 cm/s representing a 3 fold increase in comparison with the pure API. The results indicate that both solubilization and permeability were greatly enhanced when the API is complexed in the THEDES liquid form. The results obtained demonstrate the potential of THEDES to overcome the drawbacks of poorly water soluble molecules and increase bioavailability of the APIs and the possibility to develop new delivery systems.info:eu-repo/semantics/publishedVersio

Duarte, Ana Rita C.

Barreiros, Susana

Matias, Ana A.

Paiva, Alexandre

Solubility and Permeability Enhancement of active compounds: Therapeutic Deep Eutectic Systems as New Vehicles for Drug Delivery  Ana Rita C. Duarte1,2* Susana Barreiros3, Ana A. Matias4, Rui L. Reis 1,2, Alexandre Paiva3,   1 3B’s Research Group- Biomaterials, Biodegradable and Biomimetic, University of Minho, Headquarters of the European Institute of Excellence on Tissue Engineering and Regenerative Medicine, Avepark 4805-017 Barco, Guimarães, Portugal 2 ICVS/3B’s PT Government Associated Laboratory, Braga/Guimarães, Portugal 3 LAQV-REQUIMTE, Departamento de Química, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, 2829-516 Caparica, Portugal 4 Food & Health Unit, Nutraceuticals & Delivery Lab, Instituto de Biologia Experimental e Tecnológica, Oeiras, Portugal,         *Corresponding author:aduarte@dep.uminho.pt   Keywords: Therapeutic Deep Eutectic Solvents, Pharmaceuticals, Solubility, Permeability   Abstract  Therapeutic deep eutectic systems (THEDES) are here proposed as a new class of pharmaceutical active ingredient (API) whose distinctive characteristic is the fact that they are liquid at room temperature. Eutectic systems have been described in the pharmaceutical sciences as an alternative formulation able to enhance bioavailability of the API. THEDES are constituted by one active agent and a coformer or two active agents, which when combined at a particular molar ratio become liquid at room temperature. In this work, we explore the prepararation of the THEDES menthol-ibuprofen. The solubility and dissolution profile in phosphate buffer solution (pH 7.4) was performed at 37 ºC and compared with those of the pure API. Furthermore, the in vitro permeability of the liquid THEDES and the API in powder form, was evaluated using a Franz diffusion cell and caco-2 cells as model of human intestinal epithelium. The solubility of the API’s when in the THEDES system improved as much as up to 12 fold. Furthermore, for this system the permeability was calculated to be 14 x 10-5 cm/s representing a 3 fold increase in comparison with the pure API. The results indicate that both solubilization and permeability were greatly enhanced when the API is complexed in the THEDES liquid form. The results obtained demonstrate the potential of THEDES to overcome the drawbacks of poorly water soluble molecules and increase bioavailability of the APIs and the possibility to develop new delivery systems.      

Solubility and Permeability Enhancement of active compounds: Therapeutic Deep Eutectic Systems as New Vehicles for Drug Delivery

Kwan, CK

This is a repository copy of Real-time Visualisation of Actin Filaments within Native and Tissue Engineered Cartilage using SiR-Actin.White Rose Research Online URL for this paper:http://eprints.whiterose.ac.uk/111944/Version: Accepted VersionProceedings Paper:Kwan, CK, Finlay, S, Wood, D et al. (2 more authors) (2016) Real-time Visualisation of Actin Filaments within Native and Tissue Engineered Cartilage using SiR-Actin. In: TIssue Engineering Part A. TERMIS - Americas Conference & Exhibition, 11-14 Dec 2016, San Diego, USA. Mary Ann Liebert , S73-S74. https://doi.org/10.1089/ten.tea.2016.5000.abstracts(c) Mary Ann Liebert, Inc. This is an author produced version of an abstract published in Tissue Engineering Part A. Uploaded in accordance with the publisher's self-archiving policy. Final publication is available from Mary Ann Liebert, Inc., publishers at: https://doi.org/10.1089eprints@whiterose.ac.ukhttps://eprints.whiterose.ac.uk/Reuse Unless indicated otherwise, fulltext items are protected by copyright with all rights reserved. The copyright exception in section 29 of the Copyright, Designs and Patents Act 1988 allows the making of a single copy solely for the purpose of non-commercial research or private study within the limits of fair dealing. The publisher or other rights-holder may allow further reproduction and re-use of this version - refer to the White Rose Research Online record for this item. Where records identify the publisher as the copyright holder, users can verify any specific terms of use on the publisher’s website. Takedown If you consider content in White Rose Research Online to be in breach of UK law, please notify us by emailing eprints@whiterose.ac.uk including the URL of the record and the reason for the withdrawal request. Real-time Visualisation of Actin Filaments within Native and Tissue Engineered Cartilage usingSiR-ActinC. K. Kwan1, S. Finlay2, D. Wood2, B. B. Seedhom2, J. Kirkham2;1 Department of Oral Biology, Institute of Medical and Biological Engineering, Leeds, UNITEDKINGDOM.2 Department of Oral Biology, School of Dentistry, Leeds, UNITED KINGDOM.Introduction: We recently reported the use of incremental compressive loading to generate cell-scaffold constructs with high modulus and cartilage-like histology.1 Our overall aim is toimprove the efficacy of construct growth, accelerating clinical translation. We asked whether aspecific range of cellular deformation promotes optimal deposition of “quality” cartilage-likeECM in maturing constructs. Our aim was to develop a staining method for real-timevisualisation of cellular deformation of living cells within constructs.Method: Three scenarios were used for experimentation; i) Bovine synoviocytes cultured in wellplates for 7 days, ii) cartilage plugs obtained from bovine knee joints and iii) tissue engineeredconstructs generated by dynamically seeding bovine synoviocytes on non-woven polyethyleneterephthalate fibre scaffolds and cultured in chondrogenic medium for 12 weeks. Actin filamentswere stained with recently developed SiR-actin2 (0.1鳥たM) for 12h at 37°C and nuclei stained withAcridine orange (1鳥たg/mL) for 30鳥min at 37°C in DMEM/F12 medium.Results: High magnification confocal images of cell monolayers showed specific and highlyresolved staining of cytoskeletal elements and cell nuclei. 3D images of native cartilage andtissue constructs showed similar staining.Conclusion: A staining methodology was optimised using a new staining molecule that canpenetrate 3D constructs and native cartilage with good resolution. Cells remained viablethoughout. The method is suitable for real-time experiments to determine cell deformation undercompression to optimise construct maturation in the longer term.References1. Finlayet al. Tissue Eng Part C. 22. 2016.2. Lukinavičius et al. Nature Methods 11:7. 2014.

Real-time Visualisation of Actin Filaments within Native and Tissue Engineered Cartilage using SiR-Actin

https://eprints.whiterose.ac.uk/111921/1/Click-crosslinkable%20collagen%20hydrogels%20for%20cytocompatible%203D%20culture%20in%20regenerative%20medicine.pdf

Dynamic platform to recreate an osteoarthritic 3D in vitro model

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Universidade do Minho: RepositoriUM

Universidade do Minho: RepositoriUM

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White Rose Research Online

Universidade do Minho: RepositoriUM

White Rose Research Online