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.

Tüm deneyler Stanford Üniversitesi İnsan deneklerin esaslara uygun olarak yapılır ve Kurumsal Değerlendirme Kurulu protokolü onaylandı. 1. Eklem Kondrosit İzolasyonu <ol><li> Total diz protezi sırasında atılan doku kıkırdak edinin ve daha önce 8 açıklandığı gibi teşrih. </li><li> Görme pürüzsüz yüzeyler için kıkırdak örneklerinin medial veya lateral femoral kondilleri seçin ve subkondral kemik etkilemeden 4-5 mm kıkırdak biyopsi kaldırmak için bir neşter ile kıkırdak yüzeyinde kesi yapmak. </li><li> Arıtılmış kollajenaz ile O / N tedavi ile doku enzimatik ayrışma ile hücre dışı matris kondrositler izole edin. 37 ° C &#39;de, kondrosit büyüme ortamı kollajenaz II (125 U / ml) ve kollajenaz IV (160 U / ml) 1: 1&#39;lik kullanın. </li><li> Ertesi gün, 70 ile ayrışmış hücreleri ihtiva eden sindirilir doku filtremm boyut Naylon örgü gözenek. 5 dakika boyunca 600 x g&#39;de DMEM / F12 ve santrifüj 20 ml ile iki kez yıkayın. </li><li> % 10 Fetal Sığır Serumu (FBS) ile desteklenmiş DMEM / F12 içinde yeniden süspansiye, 25 ug / ml askorbat, 2 mM L-glutamin, antimikrobik maddeler, aşağıdaki 1 x 10 6 hücre / 60 mm kültür çanağı yoğunluğunda izole kondrositler Plate (100 U / ml penisilin, 100 ug / ml streptomisin ve 0.25 ug / ml fungizon). Önceki biomimetic hidrojeller kapsülleme 37 ° C&#39;de plakaları tutun ve yüksek yoğunluklu bir tek tabaka olarak birincil kondrosit kültürleri korumak. </li></ol> 2. Biyomimetik Hidrojel Fabrikasyon Not: Bu yöntem sağlar poli (etilen glikol diakrilat) (PEGDA, MW 5.000 g / mol), DPBS kondroitin sülfat-metakrilat (CS-MA) ihtiva eden bir biomimetik hidrojel sentezi. Foto-başlatıcı eklenmesi fotoaktif çapraz bağlanmasını sağlar. Nihai hidrojel bileşimi% 7 tartmak içeriyort / hacim (a / h) PEGDA bölgesinin% 3 CS-MS w / v, ve ışık başlatan a / h% 0.05. <ol><li> Metakrilat grupları ile CS polimerin işlevselleştirilmesiyle CS-MA sentezlemek. <ol><li> MES 1,952 g deiyonize su, 200 ml NaCl 5,84 g (dihidroksi 2 O) eritilmesi ile 50 mM 2-morpholinoethanesulfonic asit (MES) ve 0.5 M sodyum klorür (NaCl) tamponlu çözeltisi hazırlayın. Tamponlu çözelti içinde kondroitin sülfat, sodyum tuzu, 5 g çözündürülür. </li><li> (: EDC = 1: 2 NHS mol oranı) 0,532 g (4,62 mmol) N-hidroksisüksinimid (NHS) ve 1,771 g (9,24 mmol) 1-etil-3- (3-dimetilaminopropil) -karbodiimid hidroklorür (EDC) ilave 5 dakika boyunca çözeltisi ve bir karıştırma. </li><li> Çözeltisine ve 24 saat boyunca oda sıcaklığında reaksiyon sıcaklığı (: 2: 1: EDC: AEMA = 1 NHS molar oranı) 0,765 g (4,62 mmol) 2-aminoetil metakrilat (AEMA) ekleyin. </li><li> Diyaliz tüpüne (12-14 kDa&#39;lık bir MWCO) kullanılarak, 4 gün boyunca iyonu giderilmiş suya karşı diyaliz (diH 2 O) karışımın saflaştınlır. </li><li> P Filtre0.22 um&#39;lik bir filtreden urified çözeltisi ve kurutucu ve vakum O / N çözeltisi içeren açık tüpleri. Tüm tüpleri ışıktan korunmasını sağlamak. Nem ve ışıktan korunan -20 ° C &#39;de erimiş polimeri saklayın. </li></ol></li></ol> 3. Hücre Encapsulation <ol><li> (Hücre yüklü 3D hidrojeller yumrukluyor için) PCR film ve silindirik çubuklar Otoklav ve UV ışığı altında doku kültürü kaputu 0,2 um filtrelenmiş% 70 etanol dalma ısmarlama silindirik jel kalıp sterilize edin. </li><li> Hava kabarcıkları veya sızıntıları önlemek için boşluklar olmadan otoklavlanmış PCR film ile jel kalıbın dibine kapatılmalıdır. Steril 150 mm plaka muhafaza ediniz. </li><li> Önceki biomimetic hidrojel hücre kapsülleme günlük, kollajenaz II O / N tedavi ve kollajenaz IV çözümü ile yüksek yoğunluklu kondrosit tek tabaka ayrıştırmaları. Kollajenaz VI, her kondrosit büyüme medya için kollajenaz II için 125 U / ml ve 160 U / ml kullanın37 ° C&#39;de ilave edildi. </li><li> 50 ml&#39;lik bir tüp almak ve ilave,% 5 ağırlık / hacim (ağırlık / hacim), poli (etilen glikol diakrilat) (PEGDA, molekül ağırlığı = 5000 g / mol),% 3 w / v kondroitin sülfat-metakrilat (CS-MA) ve DPBS h ışık başlatan /% 0.05 w. Çözüm karıştırmak ve bir kenara tutmak için tüp Vortex. Işıktan tüp koruyun. </li><li> , 50 ml&#39;lik bir Falcon tüpü içine ayrışmış hücreleri toplamak 5 dakika boyunca 460 x g&#39;de santrifüj sayısı ve. Aspire tüm hücreler tüp ortam 15 x 10 6 hücre / ml&#39;lik bir yoğunlukta hücreler yeniden askıya almak için, yukarıda karışık jel maddesi ekleyin. Hava kaçınarak oluşumunu kabarcıklar ise 30 kez iyice karıştırın. </li><li> Pipet 72 özel yapılmış silindirik jel kalıbına hücre hidrojel süspansiyonunun ya da tek başına hidrojel ul ve 5 dakika için 3 mW / m 2 UV ışığı (365 nm dalga boyu) maruz kalma ile jelleşme neden. Hiçbir hücre içeriği ile bazı hidrojel çözüm hazırlayın. Gelecekteki biyokimyasal ve mekanik test için negatif kontrol olarak bu yapıları kullanın. MeasuUV yoğunluğu metre ile UV ışığın yoğunluğuna yeniden. Yoğunluğunu ayarlamak için, UV ışık kaynağı ve jelleşme sırasında hidrojel iskele arasındaki mesafeyi değiştirin. NOT: hidrojel CS içerdiğinden, aselüler katkısı sadece hücresel GAG temsil hücre yüklü hidrojellerin biyokimyasal GAG içeriğinden çıkarılır. </li><li> Kapalı kolay kabuğu için filmi kesmek için bir neşter kullanın ve dikkatlice çıkarın. Silindirik çubuklar yardımıyla, polimerleşmemiş jel ve gevşek hücreler yıkayın steril DPBS 5 ml ile bir 6 yuvalı plaka içine jel üzerinden itin. </li><li> Kültür, her oyuğa kondrosit büyüme ortamı 1,5 ml içeren 24 gözlü levhalar içine, hücre yüklü hidrojeller. Kuyuya yıkanmış hidrojeller aktarmak için tek kullanımlık spatula kullanın. </li><li> Bir canlılık / Sitotoksisite kiti kullanılarak, canlı cansız boyama 24 saat sonrası kapsülleme hücre canlılığını değerlendirmek. Kültür taze kondrosit Gro değişen 3-6 hafta süreyle hücre yüklü hidrojeller ve boş hidrojeller2 günde analizler için hasat öncesi medya wth. </li></ol> 4. RNA Ekstraksiyon ve Gen İfadesi Analizleri <ol><li> Dikkatle medya aspire ve hücre-Ladin yanı sıra boş kontrolü hidrojeller steril PBS 1X koydu. </li><li> Steril bir spatula yardımı ile, temiz bir 1.5 mi Eppendorf tüpüne her hidrojel transferi ve her tüpe Tri-reaktif madde 250 ul ilave edin. RNA izole etmek için üreticinin talimatlarına uyun. </li><li> RNA izolasyonu ve miktar kullanımı her örnekten bunun 1 mcg ve sonra High Capacity cDNA Ters Transkripsiyon Kit kullanılarak cDNA içine ters transkripsiyonu gerçekleştirin. </li><li> Nicel PCR ilgi genlerin ifadesinin incelenmesi için TaqMan Gen İfade Diziler kullanın. GAPDH dahili gen ifadesi düzeylerini normale. </li><li> PCR, aşağıdaki koşullar için bir 10 ardından urasil DNA glikosilaz önceki amplikonları inaktive etmek için 50 ° C&#39;de 2 dakika inkübasyon yer alır60, 95 ° C&#39;de inkübasyon dakika Taq polimerazı aktive etmek için. Daha sonra 95 ° C de 15 sn meydana gelen PCR&#39;nin devre döngüsü gerçekleştirmek ve 60 ° C&#39;de 1 dak. </li></ol> 5. Biyokimyasal analizler <ol><li> Her hücre hidrojel yapıları için DNA ve aşağıdaki gibi sülfatlı glikozaminoglikan (sGAG) üretimini ölçmek. </li><li> Dikkatle medya aspire ve hücre-Ladin yanı sıra boş kontrolü hidrojeller steril PBS 1X koydu. Boş bir tüp tartılır ve kağıt mendil üzerinde kurutma sonra içine hidrojel koymak ve hidrojellerin ağırlığını veya ıslak ağırlığı kaydedin. </li><li> Enzimatik sindirimi için ayrı bir 1.5 mi Eppendorf tüp -20 ° C &#39;de, her bir hidrojel dondurun ve daha sonra (GAG) (DNA) Picogreen deneyi ve Dimetil-Metilen mavisi analizi çalıştırmak için sindirilmiş ürünün bir kısım kullanın. </li><li> Hidrojeller enzimatik sindirimi için papainase çözeltisi hazırlayın. <ol><li> İlk Sodyum fosfata 7.1 g dışarı tartarak PBE tamponu hazırlamake dibazik (Na 2 HPO 4) ve Etilendiamintetraasetik asit disodyum tuzu (EDTA-Na2) 1.6 g. , DH 2 O 500 ml çözülür pH 6,5&#39;e ayarlanır ve 0.22 mm&#39;lik bir filtreden filtre saflaştırılmış çözeltisi. </li><li> PBE tamponu, 20 ml L-sistein 0,035 g çözündürülür. Çözüm ve steril papain enzimi 100 ul Filtre. </li></ol></li><li> Hazır deneyleri gerçekleştirmek için zaman, -20 ° C ila tüpleri alıp dondurulmuş hidrojellere hazır papain çözüm 300 ul ekleyin. Bir havanda jel Crush ve motor tokmak ile iyice karıştırın. </li><li> Ek papain solüsyonu ile 500 ul hacim kazandırın. Kontrol jeller için aynı prosedürü uygulayın. 16 st 3,9, 60 ° C&#39;de inkübe edin. Sindirilmiş hidrojel ihtiva eden çözelti, dolayısıyla DNA ve GAG ​​ölçümü için kullanılabilir. Üreticinin pr aşağıdaki standart olarak Lambda faj DNA ile Picogreen analizi kullanılarak DNA içeriği ölçünotocol. </li><li> 1,9-dimetilmetilen mavisi (DMMB) standart 10 olarak köpekbalığı kondroitin sülfat ile boya bağlama testi kullanılarak sülfatlı-GAG içeriği ölçmek. İlgili ıslak ağırlık GAG miktarını bölerek GAG içeriği belirler. </li></ol> 6. Mekanik Test <ol><li> 10 N yük hücresi ile donatılmış bir Instron 5944 test sistemi kullanılarak serbest basınç testleri gerçekleştirin. <ol><li> In vitro kültür istenen gün sonra, hücre-hidrojel iskele ve aselüler kontrol hidrojeller sıkıştırma testi gerçekleştirin. </li><li> Oda sıcaklığında PBS banyosu içinde örnekleri daldırın ve yükleme koşulları altında kıkırdak dokunun yaşadığı fizyolojik suşu itibaren 15,% 11,12 maksimum suşuna 1% streyn / sn bir hızda sıkıştırılması% 10-20 olduğu rapor edilmiştir 13,14. </li></ol></li><li> Üçüncü mertebeden polinom denklem kullanılarak gerilme eğrileri ve eğri uyum vs stres oluşturun. Compr belirleme% 15 suş değerlerinde eğri fit denklemden essive teğet modülü. </li></ol>

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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.

Bu protokolde bildirildiği gibi, 3D hidrojeller genellikle tek tabakalı kültürler 15 ile karşılaştı fibrocartilage hücrelerine hücre dediferansiyasyon süreci kaçınarak, kültürde kondrosit fenotipi muhafaza edebiliyoruz. Ayrıca, hidrojel yapısının chondrocytes- uzun süreli kültürleri yaş ve hastalık ile ilişkili içsel hücre özelliklerini korumaktadır elverişli bir ortam ortaya koymuştur. 3B biomimetik hidrojel kullanılması birçok avantaj vardır. İlk olarak, kondroitin sülfat dahil (CS), eklem kıkırdağının içinde bulunan başlıca bir komponent, kondroitinaz salgılayarak hidrojel matrisi bozan ve ekstra hücresel matris 5, 16 yeni sentezlenmiş kıkırdak bırakmaya hücreleri sağlar. Buna ek olarak, CS gösterilmiştir artritik eklem anti-inflamatuar özelliklere sahip. Biomimetik hidrojel kıkırdak tamir hücre dağıtımı için bir iskele malzemesi olarak kullanılabilir, ve kimyasal olarak modifiye edilebilirDaha iyi doku biyomalzeme entegrasyon 17,18 kolaylaştırmak için. PEG-CS hidrojellerin kullanımı, uzun süreli kondrositlerin kültürleri ve biyokimyasal ve mekanik özelliklerinin değerlendirilmesine olanak verir. İşte biz bu platformu kıkırdak mühendisliği için en uygun hücre tipini tanımlamak amacıyla farklılaştırılmış kondrosit çeşitli kaynaklardan karşılaştırmalı analizler için yararlı olabilir nasıl gösterir. İlginçtir, hidrojeller kapsüllü kondrositlere kendi içsel yeteneklerine göre canlı kalır ve çoğalır. Hidrojel bileşimi destekleri, aslında, sağlıklı genç ve erişkin kondrositlerin büyümesi Şekil 2&#39;de gösterildiği gibi. Açıklanan hidrojel bileşimi ve yapısı da kıkırdak doku formasyonunu teşvik glikozaminoglikan ile değerlendirilen fonksiyonel hücre dışı matris birikimi (GAG ile gösterildiği gibi ) ölçümü. Buluşun bir başka avantajı, kondrosit hidrojel yapıları olanYeni oluşan kıkırdak dokusunun mekanik özellikleri için değerlendirilebilir. Serbest basınç testi karşılaştırma için aselüler hidrojel yapılmalıdır unutmayın. Hidrojeller, aslında, bağlı CS parçalarının sertliği için temel bir sertliğe sahiptir. (Bir gerilme oranı / s% 1 at)% 5-20 serbest basınç gerilme yükleme koşulları altında kıkırdak dokusu yaşadığı fizyolojik zorlanma beri kıkırdak dokusunun 11,12 mekanik testler için uygulanabilir 10-20 olduğu rapor edilmiştir % 13,14. Mekanik test hücre yüklü ve hücresel olmayan iki hidrojellerin tepkisi, kültür son nokta değerlendirilmiştir. Tarif edilen örnekte, OA kıkırdak ihtiva eden konstruktların düşük katılıkta aksine, yetişkin ve juvenil kondrositlerin ihtiva eden konstruktların benzer bir sertliğe gözlenen, yukarıda. Hücre-hidrojel yapısının bu tür mekanik özellikleri fonksiyonel özelliklerinin değerlendirilmesine olanakHücre olgunlaşma yeteneği derinlemesine analizini veren oluşturulan doku. Sonuç olarak, kıkırdak doku üretmek için farklı bir kondrosit nüfusun potansiyel çalışma 3D biomimetik hidrojellerin kullanımı yaygın olarak uygulanabilir. Burada tarif edilen in vitro çalışmaların yanısıra hücre-yüklü yapıları in vivo nakli fizyolojik bağlamda hücre olgunlaşmasını ve rejeneratif potansiyel çalışma tasavvur edilebilir. Ek biomimetik faktörlerle hidrojel platformun bundan başka değişiklikleri de, kondrosit çoğalmasını ve olgunlaşmasını optimize etmek için tasavvur edilebilir.

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.

<table border="0" cellpadding="0" cellspacing="0">
	<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.

PEG ve CS kısımları (Şekil 1) içeren biyoaktif hidrojeller insan mafsal kondrositleri 2,3,5,7 kültür ve olgunlaşması için ideal bir platform oluşturmaktadır. Farklı yaşlarda ve hastalık durumlarından Kondrositler tarif edilen yöntem ile kültürlendi ve fenotipi, gen ekspresyonu ve oluşturulan kıkırdak dokusunun biyokimyasal ve mekanik özellikleri için analiz edilebilir. Üç kondrosit örnekleri juvenile- 6 ay, bireylerde erişkin 34 yaş ve osteoarthritic- 74 yaşında, 3D biomimetik hidrojeller kültür 3 hafta sonra hasat edilmiştir. Gen ekspresyonu, normal kondrosit, çocuk ve yetişkin hem analizleri kondrosit genleri Col2a1 ve Col6a1 ekspresyonunda bir artış göstermiştir. Col6a1 (Şekil 2) ekspresyonunu sürdürmek uygun bir biomimetik ortamda kültürlenen olmasına rağmen kondrojenik fenotipinin kaybı gösterirken Aksine, hastalıklı kondrositler Col2a1 dramatik bir azalma olduğunu gösterdi. CS-PEG hidrojeller de kondrositler çoğalmaya için dinamik bir ortam olarak hizmet PEG ve CS önceden var olan matris sindirimi ve hücre etrafındaki ve hücre dışı matris olgun kıkırdak dokusunun başlıca bileşenleri bırakır. In vitro kültür 3 hafta sonra, kondrosit genişlemesi, bu özellikler göz önüne alındığında Picogreen boyası ile DNA miktarının ölçülmesi ile tahmin edilebilir. Hücreler üç grup karşılaştırmalı analizi OA kondrositler kültür 1 gün ile karşılaştırıldığında dramatik bir düşüş sergiledi ise çocuk ve yetişkin nüfusun hücre yoğunluğu değişmeden olduğunu göstermektedir. DNA içeriğinin kaybı aynı zamanda uzun dönemli kültür (Şekil 3) sırasında mümkün hücre ölümünü destek veren kondrositler OA için değil gözlenmiştir. Salgılanmış matris kültürlerin 3 hafta sonra bitiş noktası aşamasında DMMB boya bağlama deneyi ile sülfatlanmış-GAG içeriği olarak belirlendi. GAG içeriği recor olarak hidrojel yaş ağırlık normalize edilmektedirded enzimatik sindirim ve aselüler katkı önce çıkarılır. Şekil 4&#39;te gösterildiği gibi, kondrositler 3D hidrojeller kültür 3 hafta boyunca GAG benzer bir önemli miktarda biriken. Fiziksel iskele varlığı serbest basınç deneyleri 2,3 ile numunelerin biyomekanik özelliklerinin değerlendirilmesine olanak verir. Hücresel olmayan hidrojeller basınç modüllerden bir azalma olur ise, hücreye yüklü yapılar Kültürde 3 hafta (Şekil 4) daha sonra sıkıştırma modülünü muhafaza. Fenotipik, burada açıklanan biyokimyasal ve biyomekanik analizler değerlendirilmesi ve mühendislik kıkırdak için farklı kondrosit popülasyonlarının potansiyelini anlamak için, bu nedenle yararlıdır. <img alt="figür 1" src="/files/ftp_upload/53085/53085fig1.jpg" /> 3D kondrosit cul 1. PEG-CS biomimetic hidrojeller ŞekilTure. Kondrositler poli (etilen glikol diakrilat) (PEGDA) ve kondroitin sülfat-metakrilat (CS-MA) ve ısmarlama silindirik jel kalıbına dökülmüş ihtiva eden bir karışım içinde tekrar süspanse edilir. UV ışınlarına maruz sonra, jeller kalıplardan toplanır ve hücre canlılığı, canlı cansız boyanma 24 saat sonrası kapsülleme değerlendirilir katılaştı. <a href="https://www.jove.com/files/ftp_upload/53085/53085fig1large.jpg" target="_blank">Bu rakamın büyük halini görmek için lütfen buraya tıklayınız.</a> <img alt="Şekil 2," src="/files/ftp_upload/53085/53085fig2.jpg" /> Şekil 3D biomimetik hidrojeller kültürlenmiş insan kondrositlerde kondrojenik genlerin 2. ifadesi. 3B biomimetik hidrojeller kültür 3 hafta sonra genç, yetişkin ve OA kondrositler tarafından kıkırdak belirteçleri Col2a1 ve Col6a1 nicel gen ekspresyonu. Değerler gün 1. Hata b gen ifade düzeyine normalize edilmişars ortalama ± SD temsil etmektedir. p *, &lt;iki kuyruklu Student t testi ile tespit edildiği üzere 0.05. Smeriglio ark modifiye. 2 <a href="https://www.jove.com/files/ftp_upload/53085/53085fig2large.jpg" target="_blank">Bu rakamın büyük halini görmek için lütfen buraya tıklayınız.</a> <img alt="Şekil 3," src="/files/ftp_upload/53085/53085fig3.jpg" /> 3D biomimetic Hidrojellerde kültürlü insan kondrositlerde Şekil 3. DNA içeriği. 3D biomimetic hidrojellerin kültür 3 hafta sonra çocuk, yetişkin ve OA kondrositlerinde Picogreen testi ile DNA miktar tayini. Değerler gün 1&#39;de DNA düzeyinde normalize <a href="https://www.jove.com/files/ftp_upload/53085/53085fig3large.jpg" target="_blank">edilir, bu rakamın büyük halini görmek için lütfen buraya tıklayınız.</a> <img alt="Şekil 4," src="/files/ftp_upload/53085/53085fig4.jpg" /> &lt;br /&gt; Şekil 4. GAG içerik ölçümü ve 1. günde ve 3D biomimetic hidrojeller kültür 3 hafta sonra insan kondrosit serbest basınç testi. 1. günde kondrositlerde ve kültür 3 hafta sonra DMMB deneyi ile (A) GAG miktar 3D biomimetic hidrojeller. Değerler ağırlığı (ww) ıslak normalize ve ug / g olarak ifade edilmiştir. 1. günde ve 3D biomimetic hidrojeller kültür 3 hafta sonra asellüler ve hücre yüklü hidrojellerin (B) Basınç modülü (kPa). <a href="https://www.jove.com/files/ftp_upload/53085/53085fig4large.jpg" target="_blank">Görüntülemek için tıklayınız Bu rakamın büyük bir versiyonu.</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.

Eklem Kondrosit Kültür ve Kıkırdak Üretimi için 3D hidrojel iskeleleri

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 Görüntüleme.  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.

3D parçacıklarının Üretimi için Basit Asma Bırak Hücre Kültürü Protokolü

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

Biyomühendislik

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.

3D Fibrin hazırlanması Kök Hücre Kültürü Uygulamaları için iskeleleri

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.

3-Boyutlu Hücre Kültürü için Self-raporlama iskeleleri

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.

Decellularized Akciğer Yapı iskeleleri kullanarak ESC türetilmiş Fare Havayolu Epitel Hücreleri Üretimi

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.

Deneysel ve Sonlu Eleman Protokolü Eklem Kıkırdak genelinde Nötr ve Yüklü Çözünenlerin Ulaştırma Araştırma

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.

Kıkırdak Yenileme için 3D Manyetik Kök Hücre Toplama ve Biyoreaktör Olgunlaşma

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.

Bioprinting kıkırdak ve cilt doku analogları bir roman pasif birimi teknik Bioink Precellularization için karıştırma kullanmak

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.

Gerçek zamanlı görselleştirme ve analiz nedeniyle tam olarak olduğu gibi fare kıkırdak Explants mekanik yükleme kondrosit yaralanma

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.

Decellularized matris kıkırdak elde edilen iskele imalatı

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 gözenekli selüloz Nanocomposite hidrojel iskele baskılı

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