construction-and-characterization-of-a-novel-vocal-fold-bioreactor

Construction and Characterization of a Novel Vocal Fold Bioreactor

In vitro engineering of mechanically active tissues requires the presentation of physiologically relevant mechanical conditions to cultured cells. To ...

University of Delaware

Natural resilin, the rubber-like protein that exists in specialized compartments of most arthropods, possesses excellent mechanical properties such as low stiffness, high resilience and effective energy storage. Recombinantly-engineered resilin-like polypeptides (RLPs) that possess the favorable attributes of native resilin would be attractive candidates for the modular design of biomaterials for engineering mechanically active tissues. Based on our previous success in creating a novel RLP-based hydrogel and demonstrating useful mechanical and cell-adhesive properties, we have produced a suite of new RLP-based constructs, each equipped with 12 repeats of the putative resilin consensus sequence and a single, distinct biologically active domain. This approach allows independent control over the concentrations of cell-binding, MMP-sensitive, and polysaccharide-sequestration domains in hydrogels comprising mixtures of the various RLPs. The high purity, molecular weight and correct compositions of each new polypeptide have been confirmed via high performance liquid chromatography (HPLC), sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS), and amino acid analysis. These RLP-based polypeptides exhibit largely random-coil conformation, both in solution and in the cross-linked hydrogels, as indicated by circular dichroic and infrared spectroscopic analyses. Hydrogels of various compositions, with a range of elastic moduli (1kPa to 25kPa) can be produced from these polypeptides, and the activity of the cell-binding and matrix metalloproteinase (MMP) sensitive domains was confirmed. Tris(hydroxymethyl phosphine) cross-linked RLP hydrogels were able to maintain their mechanical integrity as well as the viability of encapsulated primary human mesenchymal stem cells (MSCs). These results validate the promising properties of these RLP-based elastomeric biomaterials.

Resilin-Like Polypeptide Hydrogels Engineered for Versatile Biological Functions.

R&eacute;siline naturelle, la prot&eacute;ine caoutchouteux qui existe dans les milieux sp&eacute;cialis&eacute;s de la plupart des arthropodes, poss&egrave;de des propri&eacute;t&eacute;s m&eacute;caniques excellentes comme faible rigidit&eacute;, haute r&eacute;silience et stockage de l&#39;&eacute;nergie efficace. Inoculation-machin&eacute; comme r&eacute;siline polypeptides (RLPs) que poss&egrave;dent les attributs favorables de r&eacute;siline native serait attrayants candidats pour la conception modulaire des biomat&eacute;riaux pour l&#39;ing&eacute;nierie des tissus actifs m&eacute;caniquement. Bas&eacute;e sur nos pr&eacute;c&eacute;dents succ&egrave;s en cr&eacute;ant un roman bas&eacute; sur RLP hydrogel et d&eacute;montrant les propri&eacute;t&eacute;s utiles de m&eacute;caniques et cellule-adh&eacute;sif, nous avons produit une s&eacute;rie de nouvelles constructions de base RLP, chacune &eacute;quip&eacute;e de 12 r&eacute;p&eacute;titions de la s&eacute;quence consensus r&eacute;siline putatif et un domaine biologiquement actif unique et distinct. Cette approche permet un contr&ocirc;le ind&eacute;pendant sur les concentrations de liaison cellulaire, MMP-sensible et domaines polysaccharide-s&eacute;questration d&#39;hydrogels compos&eacute;e de m&eacute;langes des RLPs divers. Le haut degr&eacute; de puret&eacute;, de poids mol&eacute;culaire et de compositions correctes de chaque nouveau polypeptide ont &eacute;t&eacute; confirm&eacute;es par chromatographie liquide haute performance (HPLC), sur gel de polyacrylamide de sodium dodecyl sulfate (SDS-PAGE), spectrom&eacute;trie de masse de d&eacute;sorption-ionisation laser assist&eacute;e par matrice (MALDI-MS) et analyse des acides amin&eacute;s. Ces polypeptides ax&eacute;e sur le RLP pi&egrave;ce conformation de pelote al&eacute;atoire dans une large mesure, tant en solution dans les hydrogels r&eacute;ticul&eacute;s, comme indiqu&eacute; par la circulaires les analyses spectroscopiques dichro&iuml;ques et infrarouges. Hydrogels de diverses compositions, avec une gamme de modules d&#39;&eacute;lasticit&eacute; (1kPa &agrave; 25kPa) peuvent provenir de ces polypeptides, et l&#39;activit&eacute; des cellules et la matrice m&eacute;talloprot&eacute;inases (MMP) domaines sensibles a &eacute;t&eacute; confirm&eacute;e. Tris(hydroxymethyl phosphine) r&eacute;ticul&eacute;s RLP hydrogels purent conserver leur int&eacute;grit&eacute; m&eacute;canique ainsi que la viabilit&eacute; d&#39;encapsul&eacute; primaires humains cellules souches m&eacute;senchymateuses (CSM). Ces r&eacute;sultats valident les propri&eacute;t&eacute;s prometteuses de ces biomat&eacute;riaux &eacute;lastom&egrave;re RLP-bas&eacute;.

R&eacute;siline-Like Polypeptide Hydrogels machin&eacute;s pour des fonctions biologiques polyvalentes.

Naturale resilina, la gomma-come proteina che esiste in comparti specializzati della maggior parte degli artropodi, possiede eccellenti propriet&agrave; meccaniche quali bassa rigidit&agrave;, ad alta resilienza e stoccaggio di energia efficace. Recombinantly-engineered resilina-come polipeptidi (RLPs) che possiedono gli attributi favorevoli della resilina nativo sarebbe attraenti candidati per la progettazione modulare di biomateriali per l&#39;ingegneria dei tessuti meccanicamente attivi. Basato sul nostro precedente successo nella creazione di un romanzo basato su RLP hydrogel e dimostrando utili propriet&agrave; meccaniche e cella-adesivo, abbiamo prodotto una suite di nuovi costrutti di base di RLP, ciascuno dotato di 12 ripetizioni della sequenza consenso putativo resilina e un singolo dominio biologicamente attivo distinto. Questo approccio permette il controllo indipendente sulle concentrazioni di cella-associazione, MMP-sensibili e domini polisaccaride-sequestro in idrogel composto da miscele del RLPs vari. L&#39;elevata purezza, peso molecolare e corrette composizioni di ogni nuovo polipeptide sono stati confermati mediante cromatografia liquida ad alta prestazione (HPLC), sodio dodecil solfato poliacrilammide elettroforesi (SDS-PAGE), spettrometria di massa desorbimento/ionizzazione laser assistita da matrice (MALDI-MS) e analisi dell&#39;amminoacido. Questi polipeptidi RLP-based presentano conformazione in gran parte casuale-bobina, sia in soluzione che in idrogel reticolato, come indicato dalla circolare dicroiche e infrarossi analisi spettroscopiche. Idrogel di varie composizioni, con una vasta gamma di moduli elastici (1kPa a 25kPa) pu&ograve; essere prodotto da questi polipeptidi, ed &egrave; stata confermata l&#39;attivit&agrave; i domini sensibili cella-associazione e matrix metalloproteinase (MMP). Tris(hydroxymethyl phosphine) reticolato RLP idrogel sono stati in grado di mantenere la loro integrit&agrave; meccanica, nonch&eacute; la redditivit&agrave; della incapsulato primarie umane cellule staminali mesenchimali (MSCs). Questi risultati convalidano le propriet&agrave; promettente di questi biomateriali elastomerici RLP-based.

Polipeptide resilina-come gli idrogel ingegnerizzati per versatili funzioni biologiche.

We are interested in the in vitro engineering of artificial vocal fold tissues via the strategic combination of multipotent mesenchymal stem cells (MSCs), physiologically relevant mechanical stimulations, and biomimetic artificial matrices. We have constructed a vocal fold bioreactor that is capable of imposing vibratory stimulations on the cultured cells at human phonation frequencies. Separately, fibrous poly (É›-caprolactone) (PCL) scaffolds emulating the ligamentous structure of the vocal fold were prepared by electrospinning, were incorporated in the vocal fold bioreactor, and were driven into a wave-like motion in an axisymmetrical fashion by the oscillating air. MSC-laden PCL scaffolds were subjected to vibrations at 200â€‰Hz with a normal center displacement of âˆ¼40â€‰Î¼m for a total of 7 days. A continuous (CT) or a 1â€‰h-on-1â€‰h-off (OF) regime with a total dynamic culture time of 12â€‰h per day was applied. The dynamic loading did not cause any physiological trauma to the cells. Immunohistotochemical staining revealed the reinforcement of the actin filament and the enhancement of Î±5Î²1 integrin expression under selected dynamic culture conditions. Cellular expression of essential vocal fold extracellular matrix components, such as elastin, hyaluronic acid, and matrix metalloproteinase-1, was significantly elevated as compared with the static controls, and the OF regime is more conducive to matrix production than the CT vibration mode. Analyses of genes of typical fibroblast hallmarks (tenascin-C, collagen III, and procollagen I) as well as markers for MSC differentiation into nonfibroblastic lineages confirmed MSCs' adaptation of fibroblastic behaviors. Overall, the high-frequency vibratory stimulation, when combined with a synthetic fibrous scaffold, serves as a potent modulator of MSC functions. The novel bioreactor system presented here, as a versatile, yet well-controlled model, offers an in vitro platform for understanding vibration-induced mechanotransduction and for engineering of functional vocal fold tissues.

Modulating the Behaviors of Mesenchymal Stem Cells Via the Combination of High-Frequency Vibratory Stimulations and Fibrous Scaffolds.

Nous sommes int&eacute;ress&eacute;s par l&#39;ing&eacute;nierie in vitro des tissus artificiels champ vocal via la combinaison strat&eacute;gique de cellules multipotentes de souches m&eacute;senchymateuses (CSM), les stimulations m&eacute;caniques physiologiquement pertinentes et biomim&eacute;tiques matrices artificielles. Nous avons construit un bior&eacute;acteur de champ vocal qui est capable d&#39;imposer les stimulations vibrantes sur les cellules cultiv&eacute;es &agrave; des fr&eacute;quences de phonation humaine. S&eacute;par&eacute;ment, poly fibreux (&#x25B;-caprolactone) (PCL) &eacute;chafaudages imitant la structure ligamentaire du pli vocal ont &eacute;t&eacute; pr&eacute;par&eacute;s par &eacute;lectrofilage, ont &eacute;t&eacute; incorpor&eacute;es dans le bior&eacute;acteur de champ vocal et ont &eacute;t&eacute; conduits dans un mouvement ondulatoire de fa&ccedil;on axisym&eacute;trique de l&#39;air oscillant. MSC-laden PCL &eacute;chafaudages ont &eacute;t&eacute; soumis &agrave; des vibrations &agrave; 200 Hz avec un d&eacute;placement du centre normal de &sim;40 &mu;m pour un total de 7 jours. Un continu (CT) ou un 1 h contre 1 h &agrave; pied (du r&eacute;gime) avec un temps total de la culture dynamique de 12 h par jour a &eacute;t&eacute; appliqu&eacute;e. Le chargement dynamique n&#39;a pas caus&eacute; de tout traumatisme physiologique aux cellules. La coloration de Immunohistotochemical r&eacute;v&egrave;le l&#39;armature du filament d&#39;actine et l&#39;am&eacute;lioration de l&#39;expression de l&#39;int&eacute;grine &alpha;5&beta;1 dans des conditions de culture dynamique s&eacute;lectionn&eacute;. Expression cellulaire des composants de matrice extracellulaire champ vocal essentielles, telles que l&#39;&eacute;lastine, acide hyaluronique et matrice m&eacute;talloprot&eacute;inase-1, &eacute;tait significativement &eacute;lev&eacute;e par rapport aux contr&ocirc;les statiques, et le r&eacute;gime d&#39;est plus propice &agrave; la production de la matrice que le mode de vibration de CT. Analyses des g&egrave;nes des caract&eacute;ristiques typiques de fibroblastes (t&eacute;nascine-C, collag&egrave;ne III et procollag&egrave;ne je) ainsi que les marqueurs de diff&eacute;renciation de MSC en lign&eacute;es nonfibroblastic a confirm&eacute; l&#39;adaptation des MSCs de comportements fibroblastiques. Dans l&#39;ensemble, la stimulation vibratoire de haute fr&eacute;quence, lorsqu&#39;il est combin&eacute; avec un &eacute;chafaudage fibreux synth&eacute;tique, sert un puissant modulateur de fonctions MSC. Le syst&egrave;me de bior&eacute;acteur roman pr&eacute;sent&eacute; ici, comme un appareil polyvalent, mais mod&egrave;le bien contr&ocirc;l&eacute;e, offrant une plate-forme in vitro pour la m&eacute;canotransduction induite par la vibration de compr&eacute;hension et pour l&#39;ing&eacute;nierie des tissus fonctionnels champ vocal.

Modulant le comportement des cellules souches m&eacute;senchymateuses Via la combinaison de haute fr&eacute;quence vibratoires Stimulations et &eacute;chafaudages fibreuses.

Siamo interessati in ingegneria in vitro dei tessuti artificiali piega vocale tramite la combinazione strategica di multipotenti le cellule staminali mesenchimali (MSCs), fisiologicamente rilevanti stimolazioni meccaniche e matrici artificiali biomimetici. Noi abbiamo costruito un bioreattore piega vocale che &egrave; in grado di imporre stimolazioni vibranti sulle cellule coltivate a frequenze di fonazione umana. Separatamente, fibrosa poli (&#x25B;-caprolattone) (PCL) ponteggi emulando la struttura legamentosa della piega vocale sono stati preparati da elettrofilatura, furono incorporate nel bioreattore piega vocale e sono stati cacciati in un movimento ondulatorio in maniera assialsimmetrici dall&#39;aria oscillante. MSC-laden PCL ponteggi sono stati sottoposti a vibrazioni a 200 Hz con un dislocamento normale centro &sim;40 &mu;m per un totale di 7 giorni. &Egrave; stato applicato un continuo (CT) o un 1 h-a-1 h-off (del regime di) con un tempo di cultura dinamica totale di 12 ore al giorno. Il caricamento dinamico non ha causato un trauma fisiologico alle cellule. Immunohistotochemical colorazione ha rivelato il rinforzo del filamento dell&#39;actina e la valorizzazione dell&#39;espressione dell&#39;integrina &alpha;5&beta;1 sotto condizioni di coltura dinamico selezionato. Cellulare espressione dei componenti della matrice extracellulare essenziale piega vocale, come elastina, acido ialuronico e matrix metalloproteinase-1, &egrave; stato significativamente elevato rispetto i controlli statici, e di regime &egrave; pi&ugrave; favorevole alla produzione di matrice rispetto alla modalit&agrave; di vibrazione di CT. Analisi dei geni di tratti distintivi tipici dei fibroblasti (collagene tenascina-C, III e procollagene I) cos&igrave; come marcatori per la differenziazione di MSC in lignaggi nonfibroblastic confermato adattamento dei MSCs dei comportamenti fibroblastico. Nel complesso, la stimolazione ad alta frequenza vibratoria, quando combinato con un&#39;impalcatura fibrosa sintetico, serve come un potente modulatore di funzioni di MSC. Il sistema di bioreattore romanzo presentato qui, come un versatile, ancora ben controllato modello, offre una piattaforma in vitro per la comprensione delle vibrazioni indotte mechanotransduction e per l&#39;ingegneria dei tessuti funzionali piega vocale.

Modulando i comportamenti di cellule staminali mesenchimali tramite la combinazione di alta frequenza vibratorie stimolazioni e ponteggi fibrosi.

Block copolymers of poly(ethylene glycol) and poly(ε-caprolactone) (PCL) with chemically addressable functional groups were synthesized and characterized. Ring-opening polymerization of ε-caprolactone (CL) and 1,4,8-trioxaspiro-[4,6]-9-undecanone (TSU) using α-methoxy, ω-hydroxyl poly(ethylene glycol) as the initiator afforded a copolymer with cyclic ketals being randomly distributed in the hydrophobic PCL block. At an initiator/catalyst molar ratio of 10/1 and a TSU/CL weight ratio of 1/4, a ketal-carrying copolymer (ECT2-CK) with Mn of 52 kDa and a ketal content of 15 mol.% was obtained. Quantitative side-chain deacetalization revealed the reactive ketones without noticeable polymer degradation. In our study, 10 mol.% of cyclic ketals were deprotected and the ketone-containing copolymer was designated as ECT2-CO. Reaction of ECT2-CO with 2-(2-(aminooxy)acetoxy)-ethyl acrylate gave rise to an acrylated product (ECT2-AC) containing an estimated 3-5 acrylate groups per chain. UV-initiated radical polymerization of ECT2-AC in dichloromethane resulted in a crosslinked network (xECT2-AC). Thermal and morphological analyses employing differential scanning calorimetry and atomic force microscopy operated in PeakForce Tapping mode revealed the semicrystalline nature of the network, which contained stiff crystalline lamellae dispersed in a softer amorphous interstitial. Macroscopic and nanoscale mechanical characterizations showed that ECT2-CK exhibited a significantly lower modulus than PCL of a similar molecular weight. Whereas ECT2-CK undergoes a plastic deformation with a distinct yield point and a cold-drawing region, xECT2-AC exhibits a compliant, elastomeric deformation with a Young's modulus of 0.5±0.1 MPa at 37°C. When properly processed, the crosslinked network exhibited shape-memory behaviors, with shape fixity and shape recovery values close to 1 and a shape recovery time of less than 4s at 37°C. In vitro studies showed that xECT2-AC films did not induce any cytotoxic effects on the cultured mesenchymal stem cells. The crosslinkable polyester copolymers can be potentially used as tissue engineering scaffolds and minimally invasive medical devices.

Poly(ε-caprolactone)-based copolymers bearing pendant cyclic ketals and reactive acrylates for the fabrication of photocrosslinked elastomers.

Synthetic hydrogels containing covalently integrated soft and deformable drug depots capable of releasing therapeutic molecules in response to mechanical forces are attractive candidates for the treatment of degenerated tissues that are normally load bearing. Herein, radically cross-linkable block copolymer micelles (xBCM) assembled from an amphiphilic block copolymer consisting of hydrophilic poly(acrylic acid) (PAA) partially modified with 2-hydroxyethyl acrylate, and hydrophobic poly(n-butyl acryclate) (PnBA) were employed as the drug depots and the microscopic cross-linkers for the preparation of hyaluronic acid (HA)-based, hydrogels. HA hydrogels containing covalently integrated micelles (HAxBCM) were prepared by radical polymerization of glycidyl methacrylate (GMA)-modified HA (HAGMA) in the presence of xBCMs. When micelles prepared from the parent PAA-b-PnBA without any polymerizable double bonds were used, hydrogels containing physically entrapped micelles (HApBCM) were obtained. The addition of xBCMs to a HAGMA precursor solution accelerated the gelation kinetics and altered the hydrogel mechanical properties. The resultant HAxBCM gels exhibit an elastic modulus of 847 ± 43 Pa and a compressive modulus of 9.2 ± 0.7 kPa. Diffusion analysis of Nile Red (NR)-labeled xBCMs employing fluorescence correlation spectroscopy confirmed the covalent immobilization of xBCMs in HA networks. Covalent integration of dexamethasone (DEX)-loaded xBCMs in HA gels significantly reduced the initial burst release and provided sustained release over a prolonged period. Importantly, DEX release from HAxBCM gels was accelerated by intermittently applied external compression in a strain-dependent manner. Culturing macrophages in the presence of DEX-releasing HAxBCM gels significantly reduced cellular production of inflammatory cytokines. Incorporating mechano-responsive modules in synthetic matrices offers a novel strategy to harvest mechanical stress present in the healing wounds to initiate tissue repair.

Hyaluronic acid-based hydrogels containing covalently integrated drug depots: implication for controlling inflammation in mechanically stressed tissues.

Vocal fold disorders affect 3-9% of the U.S. population. Tissue engineering offers an alternative strategy for vocal fold repair. Successful engineering of vocal fold tissues requires a strategic combination of therapeutic cells, biomimetic scaffolds, and physiologically relevant mechanical and biochemical factors. Specifically, we aim to create a vocal fold-like microenvironment to coax stem cells to adopt the phenotype of vocal fold fibroblasts (VFFs). Herein, high frequency vibratory stimulations and soluble connective tissue growth factor (CTGF) were sequentially introduced to mesenchymal stem cells (MSCs) cultured on a poly(ɛ-caprolactone) (PCL)-derived microfibrous scaffold for a total of 6 days. The initial 3-day vibratory culture resulted in an increased production of hyaluronic acids (HA), tenascin-C (TNC), decorin (DCN), and matrix metalloproteinase-1 (MMP1). The subsequent 3-day CTGF treatment further enhanced the cellular production of TNC and DCN, whereas CTGF treatment alone without the vibratory preconditioning significantly promoted the synthesis of collagen I (Col 1) and sulfated glycosaminoglycans (sGAGs). The highest level of MMP1, TNC, Col III, and DCN production was found for cells being exposed to the combined vibration and CTGF treatment. Noteworthy, the vibration and CTGF elicited a differential stimulatory effect on elastin (ELN), HA synthase 1 (HAS1), and fibroblast-specific protein-1 (FSP-1). The mitogenic activity of CTGF was only elicited in naïve cells without the vibratory preconditioning. The combined treatment had profound, but opposite effects on mitogen-activated protein kinase (MAPK) pathways, Erk1/2 and p38, and the Erk1/2 pathway was critical for the observed mechano-biochemical responses. Collectively, vibratory stresses and CTGF signals cooperatively coaxed MSCs toward a VFF-like phenotype and accelerated the synthesis and remodeling of vocal fold matrices.

Zhixiang Tong

Department of Materials Science and Engineering,
Delaware Biotechnology Institute,
Department of Materials Science and Engineering, Delaware Biotechnology Institute

University of Delaware

Construction and Characterization of a Novel Vocal Fold Bioreactor

Zhixiang Tong

Department of Materials Science and Engineering,Delaware Biotechnology Institute,Department of Materials Science and Engineering, Delaware Biotechnology Institute

University of Delaware

Construction and Characterization of a Novel Vocal Fold Bioreactor

Department of Materials Science and Engineering,
Delaware Biotechnology Institute,
Department of Materials Science and Engineering, Delaware Biotechnology Institute