The authors would like to acknowledge Stanford Department of Orthopaedic Surgery and Stanford Coulter Translational Seed Grant for funding. J.H.L. would like to thank National Science Foundation Graduate Fellowship and DARE Doctoral Fellowship for support.

Tutti gli esperimenti sono stati eseguiti in conformità con le linee guida dei soggetti Stanford University Umani e approvati protocollo Institutional Review Board. Isolamento 1. articolare di condrociti <ol><li> Ottenere la cartilagine da tessuti scartati durante protesi totale di ginocchio e sezionare come descritto in precedenza 8. </li><li> Visivamente selezionare i condili femorali mediale o laterale di campioni di cartilagine per superfici lisce e rendere incisione alla superficie della cartilagine attraverso un bisturi per rimuovere 4-5 biopsie mm cartilagine senza influenzare l&#39;osso subcondrale. </li><li> Isolare condrociti dalla matrice extracellulare per dissociazione enzimatica del tessuto con il trattamento / N O con collagenasi purificata. Utilizzare rapporto 1: 1 di collagenasi II (125 U / ml) e Collagenasi IV (160 U / ml) in terreni di coltura di condrociti a 37 ° C. </li><li> Il giorno seguente, filtrare il tessuto digerito contenente le cellule dissociate attraverso un 70mm pori maglie di dimensione nylon. Lavare due volte con 20 ml di DMEM / F12 e centrifugare a 600 xg per 5 min. </li><li> Piastra le condrociti isolati ad una densità di 1 x 10 6 cellule / 60 mm piatto di coltura dopo risospensione in DMEM / F12 addizionato con 10% Fetal Bovine Serum (FBS), 25 mg / ascorbato ml, 2 mM L-glutammina, antimicrobici (100 U / ml di penicillina, 100 mg / ml di streptomicina, e 0,25 mg / ml Fungizone). Conservare le piastre a 37 ° C e mantenere le colture di condrociti primari come un monostrato alta densità prima incapsulamento in idrogel biomimetici. </li></ol> 2. Biomimetic Hydrogel Fabrication NOTA: Questo metodo permette la sintesi di un idrogel biomimetico contenente poli (etilene glicol diacrilato) (PEGDA, MW 5000 g / mole), condroitin solfato-metacrilato (CS-MA) in DPBS. L&#39;aggiunta di fotoiniziatore permette reticolazione fotoattivato. La composizione idrogel finale contiene il 7% pesaret / Volume (w / v) di PEGDA, 3% w / v di CS-MA, e 0,05% w / v di fotoiniziatore. <ol><li> Sintetizzare CS-MA per funzionalizzazione CS polimero con gruppi di metacrilato. <ol><li> Preparare la soluzione tampone di 50 mM di acido 2-morpholinoethanesulfonic (MES) e 0,5 M di cloruro di sodio (NaCl) sciogliendo 1,952 g di MES e 5,84 g di NaCl in 200 ml di acqua deionizzata (DIH 2 O). Sciogliere 5 g di sale di condroitin solfato sodico nella soluzione tamponata. </li><li> Aggiungere 0,532 g (4,62 mmol) N-idrossisuccinimmide (NHS) e 1.771 g (9.24 mmol) 1-etil-3- (3-dimetilamminopropil) cloridrato -carbodiimide (EDC) (rapporto molare di NHS: EDC = 1: 2) a la soluzione e mescolare per 5 min. </li><li> Aggiungere 0,765 g (4,62 mmol) metacrilato di 2-amminoetil (Aema) (rapporto molare di NHS: EDC: Aema = 1: 2: 1) alla soluzione e mantenere la reazione a temperatura ambiente per 24 ore. </li><li> Purificare il composto con la dialisi contro acqua deionizzata (diH 2 O) per 4 giorni usando tubo di dialisi (12-14 kDa MWCO). </li><li> Filtrare la purified soluzione attraverso un filtro di 0,22 micron e posizionare i tubi aperti contenenti la soluzione in un essiccatore sotto vuoto e O / N. Assicurarsi che tutti i tubi siano protetti dalla luce. Conservare il polimero sciolto a -20 ° C al riparo dalla luce e dall&#39;umidità. </li></ol></li></ol> Incapsulamento 3. cellulare <ol><li> Autoclave i film e cilindrici aste PCR (per punzonatura gli idrogel 3D cell-carico) e sterilizzare lo stampo gel cilindrica su misura per immersione in 0,2 micron filtrato etanolo al 70% in cappuccio coltura tissutale sotto raggi UV. </li><li> Sigillare il fondo dello stampo gel con pellicola PCR autoclave senza bolle d&#39;aria o lacune per impedire perdite. Tenerlo in una piastra sterile, 150 millimetri. </li><li> Il giorno prima di incapsulamento delle cellule in idrogel biomimetica, dissociare il condrociti monostrato ad alta densità con trattamento O / N in Collagenase II e soluzione Collagenasi IV. Utilizzare 125 U / ml per Collagenasi II e 160 U / ml per Collagenasi VI ciascuno in crescita di condrociti dei mediaa 37 ° C. </li><li> Dai tubo da 50 ml e si aggiungono 5% peso / volume (w / v) di poli (etilene glicol diacrilato) (PEGDA, MW = 5,000 g / mole), 3% w / v condroitin solfato-metacrilato (CS-MA), e 0.05% w / v fotoiniziatore in DPBS. Agitare la provetta per miscelare la soluzione e tenerlo da parte. Proteggere il tubo dalla luce. </li><li> Raccogliere le cellule dissociate in un tubo Falcon da 50 ml, contare e centrifugare a 460 xg per 5 min. Aspirare tutti i media dal tubo cellule e aggiungere il materiale sopra il gel miscelato risospendere le cellule ad una densità di 15 × 10 6 cellule / ml. Mescolare 30 volte a fondo, evitando la formazione di bolle d&#39;aria. </li><li> Dispensare 72 ml di sospensione cellulare-idrogel o da soli idrogel nello stampo gel cilindrica su misura e indurre gelificazione attraverso l&#39;esposizione alla luce UV (365 nm) a 3 mW / m 2 per 5 min. Preparare una soluzione idrogel senza contenuto della cella. Utilizzare questi costrutti come controlli negativi per il futuro i test biochimici e meccanici. Misure l&#39;intensità della luce UV con un misuratore di intensità UV. Per regolare l&#39;intensità, modificare la distanza tra la sorgente di luce UV e il ponteggio idrogel durante la gelificazione. NOTA: Dal momento che l&#39;idrogel contiene CS, il contributo acellulare viene sottratto dal contenuto GAG biochimica degli idrogel cellulari carichi di rappresentare solo il GAG cellulare. </li><li> Utilizzare un bisturi per tagliare il film per easy peel e rimuovere con cautela. Con l&#39;aiuto delle aste cilindriche, spingere fuori il gel in una piastra ben 6 con 5 ml di DPBS sterili per lavare gel polimerizzato e cellule allentati. </li><li> Culture idrogel cellulari carichi in piastre da 24 pozzetti contenenti 1,5 ml di terreni di crescita di condrociti in ogni pozzetto. Utilizzare una spatola monouso per trasferire i idrogel lavati nel pozzo. </li><li> Valutare la vitalità cellulare del morto in diretta colorazione 24 ore post-incapsulamento utilizzando un kit di sopravvivenza / citotossicità. Cultura idrogel cellulari carichi e gli idrogeli vuote per 3-6 settimane che cambiano a gro chondrocyte freschiwth dei media ogni 2 giorni prima della raccolta per le analisi. </li></ol> 4. RNA Estrazione e Gene Expression analisi <ol><li> Aspirare accuratamente la memoria e poi PBS sterile 1X sulla cellula-carico nonché gli idrogeli controllo vuote. </li><li> Con l&#39;aiuto di una spatola sterile, trasferire ogni idrogel di clean 1,5 ml provetta Eppendorf e aggiungere 250 ml di Tri-reattivo ad ogni provetta. Seguire le istruzioni del produttore per isolare l&#39;RNA. </li><li> Dopo l&#39;isolamento di RNA e quantificazione uso 1 mg di esso da ogni campione e di eseguire una trascrizione inversa in cDNA utilizzando l&#39;alta capacità cDNA corredo d&#39;inversione di trascrizione. </li><li> Per PCR quantitativa utilizzare TaqMan array di espressione genica per l&#39;esame dell&#39;espressione di geni di interesse. Normalizzare i livelli di espressione genica internamente per GAPDH. </li><li> Per le condizioni di PCR includere un 2 min di incubazione a 50 ° C per inattivare ampliconi precedenti con glicosilasi uracile-DNA, seguito da 1060; min di incubazione a 95 ° C per attivare la polimerasi Taq. Quindi eseguire cicli di PCR quaranta, costituito da 15 sec a 95 ° C, e 1 minuto a 60 ° C. </li></ol> 5. Analisi biochimiche <ol><li> Per ogni costruzioni cellulari-idrogel quantificare il DNA e glicosaminoglicani solfato (sGAG) la produzione come segue. </li><li> Aspirare accuratamente la memoria e poi PBS sterile 1X sulla cellula-carico nonché gli idrogeli controllo vuote. Pesare un tubo vuoto e mettere l&#39;idrogel all&#39;interno dopo assorbente su carta velina e registrare il peso o il peso umido degli idrogel. </li><li> Congelare ogni idrogel a -20 ° C in un tubo separato ml eppendorf 1.5 per digestione enzimatica e successivamente utilizzare una aliquota di prodotto digerita eseguire PicoGreen dosaggio (DNA) e dimetil-metilene assay blu (GAG). </li><li> Preparare la soluzione papainase per digestione enzimatica degli idrogel. <ol><li> Per prima cosa preparate il buffer PBE pesando 7,1 g di sodio phosphate bibasico (Na 2 HPO 4) e 1,6 g di acido etilendiamminotetracetico disodico sale (EDTA-Na 2). Sciogliere in 500 ml di dH 2 O, regolare il pH a 6,5 e filtrare la soluzione purificata attraverso un filtro 0.22 mm. </li><li> Sciogliere 0,035 g di L-cisteina in 20 ml di tampone PBE. Filtrare la soluzione e 100 ml di sterile papaina enzima. </li></ol></li><li> Al momento di eseguire saggi, prendere le provette da -20 ° C e aggiungere 300 microlitri della soluzione papaina pronto a idrogel congelati. Schiacciare il gel con un mortaio e pestello e mescolare bene con il pestello motore. </li><li> Portare il volume a 500 ml con soluzione papaina aggiuntivo. Eseguire la stessa procedura per i gel di controllo. Incubare a 60 ° C per 16 ore 3,9. La soluzione contenente l&#39;idrogel digerito può quindi utilizzato per il DNA e GAG ​​quantificazione. Misurare il contenuto di DNA con il saggio PicoGreen con Lambda fago DNA di serie seguente pr del produttoreotocol. </li><li> Quantificare il contenuto solfato-GAG utilizzando il blu 1,9-metilmetilene (DMMB)-dye legame test con squalo condroitin solfato di serie 10. Determinare il contenuto di GAG dividendo l&#39;importo di GAG per la corrispondente peso umido. </li></ol> 6. Prove meccaniche <ol><li> Eseguire prove di compressione non confinati usando un sistema di prova Instron 5944 dotato di una cella di carico di 10 N. <ol><li> Dopo i giorni desiderati di coltura in vitro, eseguire il test di compressione sull&#39;impalcatura cellula-idrogel e gli idrogeli controllo acellulari. </li><li> Immergere i campioni in un bagno PBS a temperatura ambiente e comprimere ad un tasso del 1% ceppo / sec per un ceppo massimo del 15% 11,12 dal momento che la sforzo fisico vissuta da tessuto cartilagineo sotto condizioni di carico è stato segnalato per essere 10-20% 13,14. </li></ol></li><li> Creare lo stress contro curve deformazione e la curva in forma con un terzo ordine un&#39;equazione polinomiale. Determinare i compressive tangente modulo dalla curva equazione forma in valori di deformazione del 15%. </li></ol>

<ol>
	<li>Roberts, S., Menage, J., Sandell, L. J., Evans, E. H., Richardson, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunohistochemical+study+of+collagen+types+I+and+II+and+procollagen+IIA+in+human+cartilage+repair+tissue+following+autologous+chondrocyte+implantation.">Immunohistochemical study of collagen types I and II and procollagen IIA in human cartilage repair tissue following autologous chondrocyte implantation.</a> Knee. 16, 398-404 (2009).</li><li>Smeriglio, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+Potential+of+Juvenile+and+Adult+Human+Articular+Chondrocytes+for+Cartilage+Tissue+Formation+in+Three-Dimensional+Biomimetic+Hydrogels.">Comparative Potential of Juvenile and Adult Human Articular Chondrocytes for Cartilage Tissue Formation in Three-Dimensional Biomimetic Hydrogels.</a> Tissue engineering. Part A. , (2014).</li><li>Lai, J. H., Kajiyama, G., Smith, R. L., Maloney, W., Yang, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stem+cells+catalyze+cartilage+formation+by+neonatal+articular+chondrocytes+in+3D+biomimetic+hydrogels.">Stem cells catalyze cartilage formation by neonatal articular chondrocytes in 3D biomimetic hydrogels.</a> Scientific reports. 3, 3553 (2013).</li><li>Taipale, J., Keski-Oja, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Growth+factors+in+the+extracellular+matrix.">Growth factors in the extracellular matrix.</a> FASEB journal : official publication of the Federation of American Societies for Experimental Biology. 11, 51-59 (1997).</li><li>Varghese, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chondroitin+sulfate+based+niches+for+chondrogenic+differentiation+of+mesenchymal+stem+cells.">Chondroitin sulfate based niches for chondrogenic differentiation of mesenchymal stem cells.</a> Matrix Biol. 27, 12-21 (2008).</li><li>Park, J. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+matrix+stiffness+on+the+differentiation+of+mesenchymal+stem+cells+in+response+to+TGF-beta.">The effect of matrix stiffness on the differentiation of mesenchymal stem cells in response to TGF-beta.</a> Biomaterials. 32, 3921-3930 (2011).</li><li>Wang, T., Lai, J. H., Han, L. H., Tong, X., Yang, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chondrogenic+Differentiation+of+Adipose-Derived+Stromal+Cells+in+Combinatorial+Hydrogels+Containing+Cartilage+Matrix+Proteins+with+Decoupled+Mechanical+Stiffness.">Chondrogenic Differentiation of Adipose-Derived Stromal Cells in Combinatorial Hydrogels Containing Cartilage Matrix Proteins with Decoupled Mechanical Stiffness.</a> Tissue engineering. Part A. , (2014).</li><li>Smith, R. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+intermittent+hydrostatic+pressure+and+BMP-2+on+osteoarthritic+human+chondrocyte+metabolism+in+vitro.">Effects of intermittent hydrostatic pressure and BMP-2 on osteoarthritic human chondrocyte metabolism in vitro.</a> J Orthop Res. 29, 361-368 (2011).</li><li>Buschmann, M. D., Gluzband, Y. A., Grodzinsky, A. J., Kimura, J. H., Hunziker, E. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chondrocytes+in+agarose+culture+synthesize+a+mechanically+functional+extracellular+matrix.">Chondrocytes in agarose culture synthesize a mechanically functional extracellular matrix.</a> J Orthop Res. 10, 745-758 (1992).</li><li>Farndale, R. W., Buttle, D. J., Barrett, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+quantitation+and+discrimination+of+sulphated+glycosaminoglycans+by+use+of+dimethylmethylene+blue.">Improved quantitation and discrimination of sulphated glycosaminoglycans by use of dimethylmethylene blue.</a> Biochim Biophys Acta. 883, 173-177 (1986).</li><li>Lai, J. H., Levenston, M. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Meniscus+and+cartilage+exhibit+distinct+intra-tissue+strain+distributions+under+unconfined+compression.">Meniscus and cartilage exhibit distinct intra-tissue strain distributions under unconfined compression.</a> Osteoarthritis and cartilage / OARS, Osteoarthritis Research Society. 18, 1291-1299 (2010).</li><li>Li, L. P., Buschmann, M. D., Shirazi-Adl, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Strain-rate+dependent+stiffness+of+articular+cartilage+in+unconfined+compression.">Strain-rate dependent stiffness of articular cartilage in unconfined compression.</a> Journal of biomechanical engineering. 125, 161-168 (2003).</li><li>Armstrong, C. G., Bahrani, A. S., Gardner, D. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vitro+measurement+of+articular+cartilage+deformations+in+the+intact+human+hip+joint+under+load.">In vitro measurement of articular cartilage deformations in the intact human hip joint under load.</a> The Journal of bone and joint surgery. American. 61, 744-755 (1979).</li><li>Macirowski, T., Tepic, S., Mann, R. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cartilage+stresses+in+the+human+hip+joint.">Cartilage stresses in the human hip joint.</a> Journal of biomechanical engineering. 116, 10-18 (1994).</li><li>Benya, P. D., Shaffer, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dedifferentiated+chondrocytes+reexpress+the+differentiated+collagen+phenotype+when+cultured+in+agarose+gels.">Dedifferentiated chondrocytes reexpress the differentiated collagen phenotype when cultured in agarose gels.</a> Cell. 30, 215-224 (1982).</li><li>Buckwalter, J. A., Mankin, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Articular+cartilage:+tissue+design+and+chondrocyte-matrix+interactions.">Articular cartilage: tissue design and chondrocyte-matrix interactions.</a> Instructional course lectures. 47, 477-486 (1998).</li><li>Wang, D. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multifunctional+chondroitin+sulphate+for+cartilage+tissue-biomaterial+integration.">Multifunctional chondroitin sulphate for cartilage tissue-biomaterial integration.</a> Nat Mater. 6, 385-392 (2007).</li><li>Simson, J. A., Strehin, I. A., Allen, B. W., Elisseeff, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bonding+and+fusion+of+meniscus+fibrocartilage+using+a+novel+chondroitin+sulfate+bone+marrow+tissue+adhesive.">Bonding and fusion of meniscus fibrocartilage using a novel chondroitin sulfate bone marrow tissue adhesive.</a> Tissue engineering. Part A. 19, 1843-1851 (2013).</li></ol>

The authors have nothing to disclose.

Come riportato in questo protocollo, gli idrogel 3D sono in grado di mantenere condrociti fenotipo nella cultura, evitando il processo di de-differenziazione delle cellule in cellule fibrocartilagine riscontrate in genere con colture monostrato 15. Inoltre, le colture a lungo termine di idrogel costrutto chondrocytes- rivelato un ambiente favorevole che mantiene le caratteristiche intrinseche delle cellule associate con l&#39;età e la malattia. L&#39;uso di un idrogel biomimetico 3D ha diversi vantaggi. In primo luogo, l&#39;inclusione di condroitin solfato (CS), uno dei principali componenti trovati nella cartilagine articolare, attivare le cellule di degradare la matrice idrogel secernendo condroitinasi e stabilire la cartilagine nuova sintesi extracellulare matrice 5, 16. Inoltre, CS è stato dimostrato per avere proprietà anti-infiammatorie nel giunto artritico. L&#39;idrogel biomimetico può anche essere usato come materiale per la consegna ponteggio cella riparazione della cartilagine, e può essere modificato chimicamenteper facilitare una migliore integrazione dei tessuti 17,18-biomateriale. L&#39;uso degli idrogel PEG-CS consente colture a lungo termine di condrociti e la valutazione delle proprietà biochimiche e meccaniche. Qui mostriamo come questa piattaforma può essere utile per le analisi comparative delle varie fonti di condrociti differenziati al fine di definire il tipo cellulare ottimale per l&#39;ingegneria cartilagine. È interessante notare che, condrociti incapsulate in idrogel rimangono vitali e proliferano in base alle loro capacità intrinseche. I supporti composizione idrogel, infatti, la crescita di giovani e adulti condrociti sani come mostrato in Figura 2. La composizione e la struttura degli idrogel descritti promuove anche la formazione di tessuto cartilagineo come indicato dalla deposizione di matrice extracellulare funzionale valutata glicosaminoglicani (GAG ) quantificazione. Un ulteriore vantaggio è che i costrutti chondrocyte-idrogelpuò essere valutata per le proprietà meccaniche del tessuto cartilagineo neoformato. Si noti che il test di compressione non confinato dovrebbe essere eseguita sul idrogel acellulare per il confronto. Gli idrogeli, infatti, hanno una rigidità intrinseca dovuta alla rigidità delle porzioni CS. Ceppo compressione unconfined del 5-20% (a velocità di deformazione 1% / s) può essere applicato per la prove meccaniche della cartilagine tessuto 11,12 in quanto il ceppo fisiologico sperimentato da tessuto cartilagineo sotto condizioni di carico è stato segnalato per essere 10-20 13,14%. La risposta delle cellule sia carichi e acellulari idrogel a prove meccaniche è stata valutata al punto finale cultura. Nell&#39;esempio descritto sopra osservato una rigidità paragonabile dei costrutti contenenti adulti e giovanili condrociti in contrasto con la rigidità inferiore dei costrutti contenenti condrociti OA. Tali proprietà meccaniche del costrutto cellule idrogel permettono la valutazione delle proprietà funzionali deltessuto formata dando una analisi approfondita della capacità maturazione delle cellule. In conclusione, l&#39;utilizzo degli idrogel biomimetici 3D per studiare il potenziale della popolazione condrociti diverso per generare tessuto cartilagineo può essere ampiamente applicato. Oltre agli studi in vitro qui descritti, trapianto in vivo dei costrutti cellulari carichi può essere prevista per studiare la maturazione delle cellule e potenziale rigenerativo nel contesto fisiologico. Ulteriori modifiche della piattaforma idrogel con ulteriori fattori biomimetici possono anche essere immaginato per ottimizzare la proliferazione di condrociti e maturazione.

With its limited self-repair potential, human articular cartilage undergoes frequent irreversible damages. Extensive efforts are currently focused on the development of efficient cell-based approaches for treatment of articular cartilage injuries. The success of these cell-based therapies is highly dependent on the selection of an optimal cell source and the maintenance of its regenerative potential. Chondrocytes are a common cell source for cartilage repair, but they are limited in supply and can de-differentiate during in vitro expansion in 2D monolayer culture thereby limiting their generation of hyaline cartilage 1. 
The aim of this protocol is to establish a 3-dimensional hydrogel platform for an in vitro comparative study of human chondrocytes from different ages and disease state. Unlike conventional two-dimensional (2D) culture, three-dimensional (3D) hydrogels allow chondrocytes to maintain their morphology and phenotype and provides a physiologically relevant environment enabling chondrocytes to produce cartilage tissue 2,3. In addition to providing a 3D physical structure for chondrocyte culture, hydrogels mimic the function of native cartilage extracellular matrix (ECM). Specifically, the inclusion of chondroitin sulfate methacrylate provides a potential reservoir for secreted paracrine factors 4 and enables cell-mediated degradation and matrix turnover 5. Although many 3D hydrogel culture systems have been utilized widely in various studies including agarose and alginate gels, we have used a biomimetic 3D culture system that has some distinct advantages for chondrocyte culture. Chondroitin sulfate (CS) is an abundant component in articular cartilage and the PEG-CS hydrogels have been shown to maintain and even enhance chondrogenic phenotype and facilitate cell-mediated matrix degradation and turnover 2,5. In addition, the mechanical properties of the hydrogel scaffold can be easily modulated by changing concentration of PEG and hence can be utilized to further enhance the regeneration potential of chondrocytes or a related cell type 6,7. PEG/CSMA is also biocompatible and hence has the potential for a direct clinical application in cartilage defects for example. The limitation for this system is its complexity and the use of photopolymerization that can potentially affect cell viability as compared to simpler systems like agarose, however the advantages for the chondrocyte culture outweigh the potential limitations.
The 3D hydrogel culture is compatible with conventional assay for evaluation of cell phenotype (gene expression, protein immunostaining) and functional outcome (quantification of cartilage matrix production, mechanical testing). This favorable 3D environment was tested to compare the tissue regeneration potential of human chondrocytes from three different aged populations in long-term 3D cultures. 
The outcomes were evaluated via both phenotypic and functional assays. Juvenile, adult and OA chondrocytes showed differential responses in the 3D biomimetic hydrogel culture. After 3 and 6 weeks, chondrogenic gene expression was upregulated in juvenile and adult chondrocytes but was downregulated in OA chondrocytes. Deposition of cartilage tissue components including aggrecan, type II collagen, and glycosaminoglycan (GAG) was high for juvenile and adult chondrocytes but not for OA chondrocytes. The compressive moduli of the resulting cartilage constructs also exhibited similar trends. In conclusion, both juvenile and adult chondrocytes exhibited chondrogenic and cartilage matrix disposition up to 6 weeks of 3D culture in hydrogels. In contrast, osteoarthritic chondrocytes revealed a loss of cartilage phenotype and minimal ability to generate robust cartilage.

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	<tbody>
		<tr>
			<td>juvenile chondrocytes (Clonetics&#8482; Normal Human Chondrocyte Cell System )</td>
			<td>Lonza</td>
			<td>CC-2550</td>
			<td></td>
		</tr>
		<tr>
			<td>adult chondrocytes (Clonetics&#8482; Normal Human Chondrocyte Cell System)</td>
			<td>Lonza</td>
			<td>CC-2550</td>
			<td></td>
		</tr>
		<tr>
			<td>poly(ethylene glycol diacrylate)</td>
			<td>Laysan Bio</td>
			<td>ACRL-PEG-ACRL-1000-1g</td>
			<td></td>
		</tr>
		<tr>
			<td>2-morpholinoethanesulfonic acid</td>
			<td>Sigma</td>
			<td>M5287</td>
			<td></td>
		</tr>
		<tr>
			<td>photoinitiator</td>
			<td>Irgacure&#160;</td>
			<td align="right">2959</td>
			<td></td>
		</tr>
		<tr>
			<td>sodium chloride&#160;</td>
			<td>Sigma</td>
			<td>S9888</td>
			<td></td>
		</tr>
		<tr>
			<td>chondroitin sulfate sodium salt&#160;</td>
			<td>Sigma</td>
			<td>C9819</td>
			<td></td>
		</tr>
		<tr>
			<td>N-hydroxysuccinimide&#160;</td>
			<td>Sigma</td>
			<td align="right">130672</td>
			<td></td>
		</tr>
		<tr>
			<td>1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride</td>
			<td>Sigma</td>
			<td>E1769</td>
			<td></td>
		</tr>
		<tr>
			<td>2-aminoethyl methacrylate</td>
			<td>Sigma</td>
			<td align="right">516155</td>
			<td></td>
		</tr>
		<tr>
			<td>dialysis tubing</td>
			<td>Spectrum Laboratories&#160;</td>
			<td align="right">132700</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase 2</td>
			<td>Worthington Biochemical&#160;</td>
			<td>LS004177</td>
			<td></td>
		</tr>
		<tr>
			<td>Collagenase 4</td>
			<td>Worthington Biochemical&#160;</td>
			<td>LS004189</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM/F12 media</td>
			<td>HyClone, Thermo Scientific&#160;</td>
			<td>SH3002301&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>live/dead assay</td>
			<td>Life Technologies</td>
			<td><a href="https://www.lifetechnologies.com/order/catalog/product/L3224">L3224</a></td>
			<td></td>
		</tr>
		<tr>
			<td>Tri reagent</td>
			<td>Life Technologies</td>
			<td>AM9738</td>
			<td></td>
		</tr>
		<tr>
			<td>Quant-iT&#8482; PicoGreen&#174; dsDNA Assay Kit</td>
			<td>Invitrogen</td>
			<td><a href="https://www.lifetechnologies.com/order/catalog/product/P11496">P11496</a></td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium phosphate dibasic&#160;</td>
			<td>Sigma</td>
			<td>S3264</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethylenediaminetetraacetic acid disodium salt&#160;</td>
			<td>Sigma</td>
			<td>E5134</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Cysteine</td>
			<td>Sigma</td>
			<td>C1276</td>
			<td></td>
		</tr>
		<tr>
			<td>1,9-dimethylmethylene blue&#160;</td>
			<td>Sigma</td>
			<td align="right">341088</td>
			<td></td>
		</tr>
		<tr>
			<td></td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Instruments</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>UV light equipment - XX-15LW Bench Lamp, 365nm</td>
			<td>UVP</td>
			<td>95-0042-07</td>
			<td></td>
		</tr>
		<tr>
			<td>Instron 5944 testing system&#160;</td>
			<td>Instron Corporation</td>
			<td>E5940</td>
			<td></td>
		</tr>
	</tbody>
</table>

3d hydrogel scaffolds for articular chondrocyte culture and cartilage generation

Human articular cartilage is highly susceptible to damage and has limited self-repair and regeneration potential. Cell-based strategies to engineer cartilage tissue offer a promising solution to repair articular cartilage. To select the optimal cell source for tissue repair, it is important to develop an appropriate culture platform to systematically examine the biological and biomechanical differences in the tissue-engineered cartilage by different cell sources. Here we applied a three-dimensional (3D) biomimetic hydrogel culture platform to systematically examine cartilage regeneration potential of juvenile, adult, and osteoarthritic (OA) chondrocytes. The 3D biomimetic hydrogel consisted of synthetic component poly(ethylene glycol) and bioactive component chondroitin sulfate, which provides a physiologically relevant microenvironment for in vitro culture of chondrocytes. In addition, the scaffold may be potentially used for cell delivery for cartilage repair in vivo. Cartilage tissue engineered in the scaffold can be evaluated using quantitative gene expression, immunofluorescence staining, biochemical assays, and mechanical testing. Utilizing these outcomes, we were able to characterize the differential regenerative potential of chondrocytes of varying age, both at the gene expression level and in the biochemical and biomechanical properties of the engineered cartilage tissue. The 3D culture model could be applied to investigate the molecular and functional differences among chondrocytes and progenitor cells from different stages of normal or aberrant development.

Idrogel bioattivi contenenti PEG e frazioni CS (Figura 1) rappresentano una piattaforma ideale per la cultura e la maturazione dei condrociti articolari umani 2,3,5,7. I condrociti di diverse epoche e stati patologici possono essere coltivati ​​con il metodo descritto ed analizzati per la loro fenotipo, espressione genica e proprietà biochimiche e meccaniche del tessuto cartilagineo generato. Tre campioni chondrocyte, juvenile- 6 mesi, adulto-34 anni e osteoarthritic- 74 anni, sono state raccolte dopo 3 settimane di coltura in 3D idrogel biomimetici. Analisi dell&#39;espressione genica di condrociti normali, sia giovani che adulti, hanno mostrato un aumento dell&#39;espressione dei geni COL2A1 e COL6A1 condrociti. Al contrario, condrociti malate mostrato una diminuzione drammatica COL2A1 mantenendo l&#39;espressione di Col6a1 (Figura 2) mostra una perdita di fenotipo condrogenica pur essendo coltivate in un ambiente biomimetico favorevole. Gli idrogel CS-PEG servono anche come un ambiente dinamico per i condrociti a proliferare, digerire la matrice preesistente di PEG e CS e depositano la loro matrice pericellulare e extracellulare, i principali componenti del tessuto cartilagineo maturo. Alla luce di queste capacità, espansione di condrociti dopo 3 settimane di coltura in vitro può essere stimato per la quantificazione del DNA con il colorante PicoGreen. Analisi comparativa dei tre gruppi di cellule mostrano che la densità cellulare di giovani e adulti popolazioni è rimasto invariato mentre condrociti OA hanno mostrato una diminuzione drammatica rispetto al giorno 1 di cultura. Stata anche osservata una perdita del contenuto di DNA per OA ma non altre condrociti suggerendo possibile morte delle cellule durante la coltura a lungo termine (Figura 3). La matrice secreta è stato quantificato come contenuto solfato-GAG per DMMB test-dye vincolante nella fase punto finale dopo 3 settimane di culture. Contenuto GAG è normalizzata per il peso umido del idrogel come recorded prima della digestione enzimatica e il contributo acellulare viene sottratto. Come mostrato in figura 4, condrociti depositato un&#39;analoga quantità significativa di GAG per 3 settimane di coltura in idrogel 3D. La presenza di un ponteggio fisica consente la valutazione delle proprietà biomeccaniche dei campioni attraverso unconfined prove di compressione 2,3. Mentre idrogel acellulari subiscono una diminuzione della moduli compressione, costrutti cellulari carichi mantenuti i moduli di compressione dopo 3 settimane in coltura (Figura 4). Il fenotipica, analisi biochimiche e biomeccaniche qui descritte sono quindi utili per valutare e comprendere le potenzialità di diverse popolazioni di condrociti per l&#39;ingegneria cartilagine. <img alt="Figura 1" src="/files/ftp_upload/53085/53085fig1.jpg" /> Figura 1. PEG-CS idrogel biomimetici per 3D condrociti cultura. condrociti sono risospesi in una miscela contenente poli (glicole etilenico diacrilato) (PEGDA) e condroitin solfato-metacrilato (CS-MA) e colato nello stampo gel cilindrica su misura. Dopo l&#39;esposizione ai raggi UV, solidificati gel sono raccolti dagli stampi e vitalità cellulare è valutata mediante colorazione morto dal vivo 24 ore post-incapsulamento. <a href="https://www.jove.com/files/ftp_upload/53085/53085fig1large.jpg" target="_blank">Cliccate qui per vedere una versione più grande di questa figura.</a> <img alt="Figura 2" src="/files/ftp_upload/53085/53085fig2.jpg" /> Figura 2. Espressione dei geni condrogeniche in condrociti umani coltivati ​​in 3D idrogel biomimetici. Espressione genica quantitativa di marker cartilagine COL2A1 e Col6a1 da giovani, adulti e OA condrociti dopo 3 settimane di coltura in 3D idrogel biomimetici. I valori sono normalizzati a livello di espressione genica a giorno 1. Errore bars rappresentano media ± SD. * p &lt;0,05 come determinato da un test t Student a due code. Modificato da Smeriglio e coll. 2 <a href="https://www.jove.com/files/ftp_upload/53085/53085fig2large.jpg" target="_blank">Cliccate qui per vedere una versione più grande di questa figura.</a> <img alt="Figura 3" src="/files/ftp_upload/53085/53085fig3.jpg" /> Figura 3. contenuto di DNA in condrociti umani coltivati ​​in idrogel biomimetici 3D. Quantificazione del DNA da PicoGreen test in giovani, adulti e OA condrociti dopo 3 settimane di coltura in 3D idrogel biomimetici. I valori sono normalizzati a livello del DNA a giorno 1. <a href="https://www.jove.com/files/ftp_upload/53085/53085fig3large.jpg" target="_blank">Clicca qui per vedere una versione più grande di questa figura.</a> <img alt="Figura 4" src="/files/ftp_upload/53085/53085fig4.jpg" /> &lt;br /&gt; Figura 4. Misurazione del contenuto di GAG e test di compressione non confinato di condrociti umani in giorno 1 e dopo 3 settimane di cultura in 3D idrogel biomimetici. (A) GAG quantificazione mediante test DMMB in condrociti al giorno 1 e dopo 3 settimane di cultura idrogel biomimetici 3D. I valori sono normalizzati a bagnare peso (ww) ed espressi in mg / g. (B) modulo di compressione (kPa) di acellulari e cellulari carichi di idrogel a giorno 1 e dopo 3 settimane di cultura in 3D idrogel biomimetici. <a href="https://www.jove.com/files/ftp_upload/53085/53085fig4large.jpg" target="_blank">Clicca qui per vedere una versione più grande di questa figura.</a>

Watch this Scientific Journal Video about 3D Hydrogel Scaffolds for Articular Chondrocyte Culture and Cartilage Generation at JoVE.com

3D Hydrogel Scaffolds for Articular Chondrocyte Culture and Cartilage Generation

Cartilage repair represents an unmet medical challenge and cell-based approaches to engineer human articular cartilage are a promising solution. Here, we describe three-dimensional (3D) biomimetic hydrogels as an ideal tool for the expansion and maturation of human articular chondrocytes.

3D Hydrogel Ponteggi per articolare condrociti, Cultura e Cartilagine Generation

Human articular cartilage is highly susceptible to damage and has limited self-repair and regeneration potential. Cell-based strategies to engineer ...

3d-hydrogel-scaffolds-for-articular-chondrocyte-culture-cartilage

Research

JoVE Journal

Bioengineering

19.8K Views.  Stanford University. Cartilage repair represents an unmet medical challenge and cell-based approaches to engineer human articular cartilage are a promising solution. Here, we describe three-dimensional (3D) biomimetic hydrogels as an ideal tool for the expansion and maturation of human articular chondrocytes. 

Video: 3D Hydrogel Scaffolds for Articular Chondrocyte Culture and Cartilage Generation

A Simple Hanging Drop Cell Culture Protocol for Generation of 3D Spheroids

Studies of cell-cell cohesion and cell-substratum adhesion have historically been performed on monolayer cultures adherent to rigid substrates. Cells within a tissue, however, are typically encased within a closely packed tissue mass in which cells establish intimate connections with many near-neighbors and with extracellular matrix components. Accordingly, the chemical milieu and physical forces experienced by cells within a 3D tissue are fundamentally different than those experienced by cells grown in monolayer culture. This has been shown to markedly impact cellular morphology and signaling. Several methods have been devised to generate 3D cell cultures including encapsulation of cells in collagen gels1or in biomaterial scaffolds2. Such methods, while useful, do not recapitulate the intimate direct cell-cell adhesion architecture found in normal tissues. Rather, they more closely approximate culture systems in which single cells are loosely dispersed within a 3D meshwork of ECM products. Here, we describe a simple method in which cells are placed in hanging drop culture and incubated under physiological conditions until they form true 3D spheroids in which cells are in direct contact with each other and with extracellular matrix components. The method requires no specialized equipment and can be adapted to include addition of any biological agent in very small quantities that may be of interest in elucidating effects on cell-cell or cell-ECM interaction. The method can also be used to co-culture two (or more) different cell populations so as to elucidate the role of cell-cell or cell-ECM interactions in specifying spatial relationships between cells. Cell-cell cohesion and cell-ECM adhesion are the cornerstones of studies of embryonic development, tumor-stromal cell interaction in malignant invasion, wound healing, and for applications to tissue engineering. This simple method will provide a means of generating tissue-like cellular aggregates for measurement of biomechanical properties or for molecular and biochemical analysis in a physiologically relevant model.

We describe a simple, rapid method of generating 3D tissue-like spheroids and their potential application to quantify differences in cell-cell interactions.

Un semplice Hanging goccia Cultura protocollo cellulare per la generazione di sferoidi 3D

Studies of cell-cell cohesion and cell-substratum adhesion have historically been performed on monolayer cultures adherent to rigid substrates. Cells ...

Ricerca

Bioingegneria

Preparation of 3D Fibrin Scaffolds for Stem Cell Culture Applications

Stem cells are found in naturally occurring 3D microenvironments in vivo, which are often referred to as the stem cell niche 1. Culturing stem cells inside of 3D biomaterial scaffolds provides a way to accurately mimic these microenvironments, providing an advantage over traditional 2D culture methods using polystyrene as well as a method for engineering replacement tissues 2. While 2D tissue culture polystrene has been used for the majority of cell culture experiments, 3D biomaterial scaffolds can more closely replicate the microenvironments found in vivo by enabling more accurate establishment of cell polarity in the environment and possessing biochemical and mechanical properties similar to soft tissue.3 A variety of naturally derived and synthetic biomaterial scaffolds have been investigated as 3D environments for supporting stem cell growth. While synthetic scaffolds can be synthesized to have a greater range of mechanical and chemical properties and often have greater reproducibility, natural biomaterials are often composed of proteins and polysaccharides found in the extracelluar matrix and as a result contain binding sites for cell adhesion and readily support cell culture. Fibrin scaffolds, produced by polymerizing the protein fibrinogen obtained from plasma, have been widely investigated for a variety of tissue engineering applications both in vitro and in vivo 4. Such scaffolds can be modified using a variety of methods to incorporate controlled release systems for delivering therapeutic factors 5. Previous work has shown that such scaffolds can be used to successfully culture embryonic stem cells and this scaffold-based culture system can be used to screen the effects of various growth factors on the differentiation of the stem cells seeded inside 6,7.
 This protocol details the process of polymerizing fibrin scaffolds from fibrinogen solutions using the enzymatic activity of thrombin. The process takes 2 days to complete, including an overnight dialysis step for the fibrinogen solution to remove citrates that inhibit polymerization. These detailed methods rely on fibrinogen concentrations determined to be optimal for embryonic and induced pluripotent stem cell culture. Other groups have further investigated fibrin scaffolds for a wide range of cell types and applications - demonstrating the versatility of this approach 8-12.

This work details the preparation of 3D fibrin scaffolds for culturing and differentiating plutipotent stem cells. Such scaffolds can be used to screen the effects of various biological compounds on stem cell behavior as well as modified to contain drug delivery systems.

Preparazione del 3D fibrina ponteggi per le applicazioni delle cellule staminali Cultura

Stem cells are found in naturally occurring 3D microenvironments in vivo, which are often referred to as the stem cell niche 1. Culturing stem cells ...

Self-reporting Scaffolds for 3-Dimensional Cell Culture

Culturing cells in 3D on appropriate scaffolds is thought to better mimic the in vivo microenvironment and increase cell-cell interactions. The resulting 3D cellular construct can often be more relevant to studying the molecular events and cell-cell interactions than similar experiments studied in 2D. To create effective 3D cultures with high cell viability throughout the scaffold the culture conditions such as oxygen and pH need to be carefully controlled as gradients in analyte concentration can exist throughout the 3D construct. Here we describe the methods of preparing biocompatible pH responsive sol-gel nanosensors and their incorporation into poly(lactic-co-glycolic acid) (PLGA) electrospun scaffolds along with their subsequent preparation for the culture of mammalian cells. The pH responsive scaffolds can be used as tools to determine microenvironmental pH within a 3D cellular construct. Furthermore, we detail the delivery of pH responsive nanosensors to the intracellular environment of mammalian cells whose growth was supported by electrospun PLGA scaffolds. The cytoplasmic location of the pH responsive nanosensors can be utilized to monitor intracellular pH (pHi) during ongoing experimentation.

Biocompatible pH responsive sol-gel nanosensors can be incorporated into poly(lactic-co-glycolic acid) (PLGA) electrospun scaffolds. The produced self-reporting scaffolds can be used for in&#160;situ monitoring of microenvironmental conditions whilst culturing cells upon the scaffold. This is beneficial as the 3D cellular construct can be monitored in real-time without disturbing the experiment.

Ponteggi self-reporting per coltura cellulare 3-Dimensional

Culturing cells in 3D on appropriate scaffolds is thought to better mimic the in vivo microenvironment and increase cell-cell interactions. The resulting ...

Generation of ESC-derived Mouse Airway Epithelial Cells Using Decellularized Lung Scaffolds

Lung lineage differentiation requires integration of complex environmental cues that include growth factor signaling, cell-cell interactions and cell-matrix interactions. Due to this complexity, recapitulation of lung development in vitro to promote differentiation of stem cells to lung epithelial cells has been challenging. In this protocol, decellularized lung scaffolds are used to mimic the 3-dimensional environment of the lung and generate stem cell-derived airway epithelial cells. Mouse embryonic stem cell are first differentiated to the endoderm lineage using an embryoid body (EB) culture method with activin A. Endoderm cells are then seeded onto decellularized scaffolds and cultured at air-liquid interface for up to 21 days. This technique promotes differentiation of seeded cells to functional airway epithelial cells (ciliated cells, club cells, and basal cells) without additional growth factor supplementation. This culture setup is defined, serum-free, inexpensive, and reproducible. Although there is limited contamination from non-lung endoderm lineages in culture, this protocol only generates airway epithelial populations and does not give rise to alveolar epithelial cells. Airway epithelia generated with this protocol can be used to study cell-matrix interactions during lung organogenesis and for disease modeling or drug-discovery platforms of airway-related pathologies such as cystic fibrosis.

This protocol efficiently directs mouse embryonic stem cell-derived definitive endoderm to mature airway epithelial cells. This differentiation technique uses 3-dimensional decellularized lung scaffolds to direct lung lineage specification, in a defined, serum-free culture setting.

Generazione di cellule di topo Airway epiteliali ESC-derivati ​​dall&#39;uso di decellularized Lung Ponteggi

Lung lineage differentiation requires integration of complex environmental cues that include growth factor signaling, cell-cell interactions and ...

An Experimental and Finite Element Protocol to Investigate the Transport of Neutral and Charged Solutes across Articular Cartilage

Osteoarthritis (OA) is a debilitating disease that is associated with degeneration of articular cartilage and subchondral bone. Degeneration of articular cartilage impairs its load-bearing function substantially as it experiences tremendous chemical degradation, i.e. proteoglycan loss and collagen fibril disruption. One promising way to investigate chemical damage mechanisms during OA is to expose the cartilage specimens to an external solute and monitor the diffusion of the molecules. The degree of cartilage damage (i.e. concentration and configuration of essential macromolecules) is associated with collisional energy loss of external solutes while moving across articular cartilage creates different diffusion characteristics compared to healthy cartilage. In this study, we introduce a protocol, which consists of several steps and is based on previously developed experimental micro-Computed Tomography (micro-CT) and finite element modeling. The transport of charged and uncharged iodinated molecules is first recorded using micro-CT, which is followed by applying biphasic-solute and multiphasic finite element models to obtain diffusion coefficients and fixed charge densities across cartilage zones.

We propose a protocol to investigate the transport of charged and uncharged molecules across articular cartilage with the aid of recently developed experimental and numerical methods.

Uno Sperimentale e Finite Element protocollo per indagare il trasporto di soluti neutri e carichi attraverso cartilagine articolare

Osteoarthritis (OA) is a debilitating disease that is associated with degeneration of articular cartilage and subchondral bone. Degeneration of articular ...

3D Magnetic Stem Cell Aggregation and Bioreactor Maturation for Cartilage Regeneration

Cartilage engineering remains a challenge due to the difficulties in creating an in vitro functional implant similar to the native tissue. An approach recently explored for the development of autologous replacements involves the differentiation of stem cells into chondrocytes. To initiate this chondrogenesis, a degree of compaction of the stem cells is required; hence, we demonstrated the feasibility of magnetically condensing cells, both within thick scaffolds and scaffold-free, using miniaturized magnetic field sources as cell attractors. This magnetic approach was also used to guide aggregate fusion and to build scaffold-free, organized, three-dimensional (3D) tissues several millimeters in size. In addition to having an enhanced size, the tissue formed by magnetic-driven fusion presented a significant increase in the expression of collagen II, and a similar trend was observed for aggrecan expression. As the native cartilage was subjected to forces that influenced its 3D structure, dynamic maturation was also performed. A bioreactor that provides mechanical stimuli was used to culture the magnetically seeded scaffolds over a 21-day period. Bioreactor maturation largely improved chondrogenesis into the cellularized scaffolds; the extracellular matrix obtained under these conditions was rich in collagen II and aggrecan. This work outlines the innovative potential of magnetic condensation of labeled stem cells and dynamic maturation in a bioreactor for improved chondrogenic differentiation, both scaffold-free and within polysaccharide scaffolds.

Chondrogenesis from stem cells requires fine tuning the culture conditions. Here, we present a magnetic approach for condensing cells, an essential step to initiate chondrogenesis. In addition, we show that dynamic maturation in a bioreactor applies mechanical stimulation to the cellular constructs and enhances cartilaginous extracellular matrix production.

3D Stem magnetica cellulare Aggregazione e bioreattore maturazione per la cartilagine Regeneration

Cartilage engineering remains a challenge due to the difficulties in creating an in vitro functional implant similar to the native tissue. An approach ...

Bioprinting of Cartilage and Skin Tissue Analogs Utilizing a Novel Passive Mixing Unit Technique for Bioink Precellularization

Bioprinting is a powerful technique for the rapid and reproducible fabrication of constructs for tissue engineering applications. In this study, both cartilage and skin analogs were fabricated after bioink pre-cellularization utilizing a novel passive mixing unit technique. This technique was developed with the aim to simplify the steps involved in the mixing of a cell suspension into a highly viscous bioink. The resolution of filaments deposited through bioprinting necessitates the assurance of uniformity in cell distribution prior to printing to avoid the deposition of regions without cells or retention of large cell clumps that can clog the needle. We demonstrate the ability to rapidly blend a cell suspension with a bioink prior to bioprinting of both cartilage and skin analogs. Both tissue analogs could be cultured for up to 4 weeks. Histological analysis demonstrated both cell viability and deposition of tissue specific extracellular matrix (ECM) markers such as glycosaminoglycans (GAGs) and collagen I respectively.

Cartilage and skin analogs were bioprinted using a nanocellulose-alginate based bioink. The bioinks were cellularized prior to printing via a single step passive mixing unit. The constructs were demonstrated to be uniformly cellularized, have high viability, and exhibit favorable markers of differentiation.

Multimateriali di cartilagine e pelle tessuto analoghi utilizzando un romanzo passivo unità tecnica di miscelazione per Bioink Precellularization

Bioprinting is a powerful technique for the rapid and reproducible fabrication of constructs for tissue engineering applications. In this study, both ...

Real-time Visualization and Analysis of Chondrocyte Injury Due to Mechanical Loading in Fully Intact Murine Cartilage Explants

Homeostasis of articular cartilage depends on the viability of resident cells (chondrocytes). Unfortunately, mechanical trauma can induce widespread chondrocyte death, potentially leading to irreversible breakdown of the joint and the onset of osteoarthritis. Additionally, maintenance of chondrocyte viability is important in osteochondral graft procedures for optimal surgical outcomes. We present a method to assess the spatial extent of cell injury/death on the articular surface of intact murine synovial joints after application of controlled mechanical loads or impacts. This method can be used in comparative studies to investigate the effects of different mechanical loading regimens, different environmental conditions or genetic manipulations, as well as different stages of cartilage degeneration on short- and/or long-term vulnerability of in situ articular chondrocytes. The goal of the protocol introduced in the manuscript is to assess the spatial extent of cell injury/death on the articular surface of murine synovial joints. Importantly, this method enables testing on fully intact cartilage without compromising native boundary conditions. Moreover, it allows for real-time visualization of vitally stained articular chondrocytes and single image-based analysis of cell injury induced by application of controlled static and impact loading regimens. Our representative results demonstrate that in healthy cartilage explants, the spatial extent of cell injury depends sensitively on load magnitude and impact intensity. Our method can be easily adapted to investigate the effects of different mechanical loading regimens, different environmental conditions or different genetic manipulations on the mechanical vulnerability of in situ articular chondrocytes.

We present a method to assess the spatial extent of cell injury/death on the articular surface of intact murine joints after application of controlled mechanical loads or impacts. This method can be used to investigate how osteoarthritis, genetic factors and/or different loading regimens affect the vulnerability of in situ chondrocytes.

Visualizzazione in tempo reale e analisi di condrociti lesioni dovute a carichi meccanici in espianti di cartilagine murina completamente intatta

Homeostasis of articular cartilage depends on the viability of resident cells (chondrocytes). Unfortunately, mechanical trauma can induce widespread ...

Fabrication of Decellularized Cartilage-derived Matrix Scaffolds

Osteochondral defects lack sufficient intrinsic repair capacity to regenerate functionally sound bone and cartilage tissue. To this extent, cartilage research has focused on the development of regenerative scaffolds. This article describes the development of scaffolds that are completely derived from natural cartilage extracellular matrix, coming from an equine donor. Potential applications of the scaffolds include producing allografts for cartilage repair, serving as a scaffold for osteochondral tissue engineering, and providing in vitro models to study tissue formation. By decellularizing the tissue, the donor cells are removed, but many of the natural bioactive cues are thought to be retained. The main advantage of using such a natural scaffold in comparison to a synthetically produced scaffold is that no further functionalization of polymers is required to drive osteochondral tissue regeneration. The cartilage-derived matrix scaffolds can be used for bone and cartilage tissue regeneration in both in vivo and in vitro settings.

Decellularized cartilage-derived scaffolds can be used as a scaffold to guide cartilage repair and as a means to regenerate osteochondral tissue. This paper describes the decellularization process in detail and provides suggestions to use these scaffolds in in vitro settings.

Fabbricazione di scaffold decellularizzati matrice della cartilagine-derivato

Osteochondral defects lack sufficient intrinsic repair capacity to regenerate functionally sound bone and cartilage tissue. To this extent, cartilage ...

3D Printed Porous Cellulose Nanocomposite Hydrogel Scaffolds

This work demonstrates the use of three-dimensional (3D) printing to produce porous cubic scaffolds using cellulose nanocomposite hydrogel ink, with controlled pore structure and mechanical properties. Cellulose nanocrystals (CNCs, 69.62 wt%) based hydrogel ink with matrix (sodium alginate and gelatin) was developed and 3D printed into scaffolds with uniform and gradient pore structure (110-1,100 &#181;m). The scaffolds showed compression modulus in the range of 0.20-0.45 MPa when tested in simulated in vivo conditions (in distilled water at 37 &#176;C). The pore sizes and the compression modulus of the 3D scaffolds matched with the requirements needed for cartilage regeneration applications. This work demonstrates that the consistency of the ink can be controlled by the concentration of the precursors and porosity can be controlled by the 3D printing process and both of these factors in return defines the mechanical properties of the 3D printed porous hydrogel scaffold. This process method can therefore be used to fabricate structurally and compositionally customized scaffolds according to the specific needs of patients.&#160;

The three critical steps of this protocol are i) developing the right composition and consistency of the cellulose hydrogel ink, ii) 3D printing of scaffolds into various pore structures with good shape fidelity and dimensions and iii) demonstration of the mechanical properties in simulated body conditions for cartilage regeneration.

3D stampato cellulosa porosa dell'idrogelo Nanocomposite ponteggi

This work demonstrates the use of three-dimensional (3D) printing to produce porous cubic scaffolds using cellulose nanocomposite hydrogel ink, with ...