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.

すべての実験は、スタンフォード大学の人間の被験者のガイドラインに沿って行われ、治験審査委員会のプロトコルを承認しました。 1.関節軟骨細胞の単離<ol><li>人工膝関節全置換術の際に廃棄された組織から軟骨を取得し、以前に説明したように8解剖。 </li><li>視覚滑らかな表面のための軟骨サンプルの内側または外側大腿骨顆を選択して、軟骨下骨に影響を与えることなく、4〜5ミリメ​​ートルの軟骨生検を除去するためにメスを介して軟骨の表面に切り込みを入れます。 </li><li>精製されたコラゲナーゼでO / N処理と組織の酵素解離によって細胞外マトリックスから軟骨細胞を分離します。 37℃での軟骨細胞の増殖培地にコラゲナーゼII（125 U / ml）およびコラゲナーゼIV（160 U / ml）を1：1の比を使用してください。 </li><li>翌日、70を介して分離した細胞を含む消化された組織をフィルタリングミリメートル孔径ナイロンメッシュ。 5分間600×gでDMEM / F12と遠心の20mlで2回洗浄します。 </li><li>プレート10％ウシ胎児血清（FBS）、25 / mlのアスコルビン酸、2mM L-グルタミン、抗菌剤を補充したDMEM / F12中に再懸濁以下、1×10 6細胞 / 60mm培養ディッシュの密度で単離された軟骨細胞（100 U / mlペニシリン、100μg/ mlのストレプトマイシン、および0.25 / mlのファンギゾン）。プレートを37℃で維持し、生体模倣ヒドロゲルでカプセル化の前に、高密度の単層としての一次軟骨細胞培養を維持します。 </li></ol> 2.バイオミメティックハイドロゲルの作製注：この方法は、DPBS中で、ポリ（エチレングリコールジアクリレート）（PEGDA、MW 5,000グラム/モル）、コンドロイチン硫酸 - メタクリレート（CS-MA）を含有する生体模倣ヒドロゲルの合成を可能にします。光開始剤の添加は、光活性化架橋を可能にします。最終のヒドロゲル組成物は、7％の重さが含まれW / V CS-MAの3％、および重量/体積の光開始剤の0.05％、PEGDAの（w / v）のT /体積。 <ol><li>メタクリレート基でCSポリマーを官能することにより、CS-MAを合成します。 <ol><li> MESの1.952グラムおよび脱イオン水200ml中のNaClの5.84グラム（DIH 2 O）を溶解させて緩衝50mMの2-モルホリノエタンスルホン酸（MES）の溶液及び0.5 M塩化ナトリウム（食塩）を準備します。緩衝液中のコンドロイチン硫酸ナトリウム塩を5g溶解します。 </li><li> （：EDC = 1：2 NHSのモル比）を0.532グラム（4.62ミリモル）のN-ヒドロキシスクシンイミド（NHS）および1.771グラム（9.24ミリモル）の1-エチル-3-（3-ジメチルアミノプロピル） - カルボジイミド塩酸塩（EDC）を加えます5分間溶液と、撹拌。 </li><li>溶液を24時間、室温で反応を維持する（2：1：EDC：NHS AEMA = 1のモル比）0.765グラム（4.62ミリモル）の2-アミノエチルメタクリレート（AEMA）を加えます。 </li><li>透析チューブ（12-14 kDaのMWCO）を使用して、4日間脱イオン水（DIH 2 O）に対する透析により混合物を精製します。 </li><li> Pをフィルタリング0.22μmのフィルターを通してurified解決し、デシケーター、真空O / Nでの溶液を含むオープンチューブを配置します。すべてのチューブは、光から保護されていることを確認します。光と湿気から保護-20℃で溶解したポリマーを保管してください。 </li></ol></li></ol> 3.細胞のカプセル化<ol><li> （セルを含んだ3Dハイドロゲルを打ち抜くための）PCRフィルムと円筒形ロッドをオートクレーブUV光の下で組織培養フードで0.2μmのフィルタ処理70％エタノールに浸漬して、カスタムメイドの円筒状のゲル型を滅菌します。 </li><li>漏れを防止するために、気泡や隙間なくオートクレーブPCR膜とゲル型の底部を密封します。無菌の150mmプレートに保管してください。 </li><li>前生体模倣ヒドロゲル中の細胞カプセル化する日は、コラゲナーゼIIおよびコラゲナーゼIV溶液中のO / N処理と高密度軟骨細胞単層を解離します。軟骨細胞の増殖培地に各コラゲナーゼIIおよびコラゲナーゼVI mlの160 U / 125 U / mlで使用37℃。 </li><li> 50 mlチューブに取り、5％重量/容積、ポリ（エチレングリコールジアクリレート）（PEGDA、MW = 5,000グラム/モル）（w / v）の3％重量/体積コンドロイチン硫酸 - メタクリレート（CS-MA）を追加し、そしてDPBS中のV光開始剤/ wの0.05％。溶液を混合し、それを脇に保つためにボルテックスチューブ。光からチューブを保護します。 </li><li> 50mlのファルコンチューブに解離した細胞を回収し、5分間460×gでカウントし、遠心します。吸引物は、すべてのセルチューブからメディア及び15×10 6細胞/ mlの密度で細胞を再懸濁するために上記混合ゲル材料を追加します。気泡の形成を回避しながら、徹底的に30回にそれを混ぜます。 </li><li>ピペット72カスタムメイドの円筒状のゲル型に細胞ヒドロゲル懸濁液又は単独のヒドロゲルのμL、5分間3ミリワット/ m 2のUV光（波長365nm）への暴露によりゲル化を誘導します。無細胞内容のいくつかのヒドロゲル溶液を準備します。将来の生化学的および機械的試験のための陰性対照として、これらの構築物を使用してください。 Measu紫外線強度計でUV光の強度を再。強度を調整するには、UV光源とゲル中のヒドロゲル足場の間の距離を変更します。 注：ヒドロゲルは、CSが含まれているため、無細胞の寄与は細胞のGAGのみを表現するために、細胞を含んだヒドロゲルの生化学GAG含有量から差し引かれます。 </li><li>オフイージーピール用のフィルムをカットするメスを使用して、慎重に取り外し。円筒状の棒の助けを借りて、未重合ゲルとゆるい細胞を洗い流し、無菌DPBS 5mlで6ウェルプレートにゲルを押し出します。 </li><li>培養物を各ウェルに軟骨細胞増殖培地1.5 mlを含有する24ウェルプレート中の細胞を含んだハイドロゲル。ウェルに洗浄ヒドロゲルを転送するために使い捨てへらを使用してください。 </li><li>生存性/細胞毒性キットを使ってライブ死んで染色後24時間カプセル化することによって、細胞の生存率を評価します。文化新鮮な軟骨細胞GROへの変更3-6週間のセルを含んだヒドロゲルおよび空のヒドロゲル2日ごとに解析のために回収する前にメディアWTH。 </li></ol> 4. RNA抽出および遺伝子発現解析<ol><li>慎重にメディアを吸引し、細胞を含んだだけでなく、空の制御ヒドロゲルに滅菌PBS 1Xを置きます。 </li><li>無菌のへらの助けを借りて、きれいな1.5mlのエッペンドルフチュー​​ブに各ヒドロゲルを転送し、各チューブにトライ試薬の250μlを添加します。 RNAを単離するために、メーカーの指示に従ってください。 </li><li>各サンプルからのRNAの単離および定量化の使用後は、それを1μgと高容量cDNAを逆転写キットを用いてcDNAに逆転写を行います。 </li><li>定量的PCRのために興味のある遺伝子の発現の検査のためのTaqMan遺伝子発現アレイを使用しています。 GAPDHの内部遺伝子発現レベルを正規化します。 </li><li> PCR条件のために10に続くウラシルDNAグリコシラーゼを用いた以前のアンプリコンを、不活性化するために50℃で2分間のインキュベーションを含みます60; 95℃でのインキュベーションminのTaqポリメラーゼを活性化します。その後、95℃で15秒間からなる40サイクルのPCRを行い、60℃で1分間。 </li></ol> 5.生化学分析<ol><li>各セルヒドロゲルの構築のためのDNAと以下のように硫酸化グリコサミノグリカン（sGAG）の生産を定量化します。 </li><li>慎重にメディアを吸引し、細胞を含んだだけでなく、空の制御ヒドロゲルに滅菌PBS 1Xを置きます。空のチューブを計量し、ティッシュペーパーの上にブロッティングした後、内部にヒドロゲルを入れて、ヒドロゲルの重量または湿重量を記録します。 </li><li>酵素消化のために別々の1.5mlのエッペンドルフチュー​​ブ中で-20℃で各ハイドロゲルを凍結し、後で（GAG用）（DNA用）ピコグリーンアッセイおよびジメチルメチレンブルーアッセイを実行するために、消化産物の一部を使用しています。 </li><li>ヒドロゲルの酵素消化のためpapainase溶液を調製します。 <ol><li>まずナトリウムphosphatの7.1グラムを計量することによりPBEバッファを用意Eの二塩基性（ の Na 2 HPO 4）、エチレンジアミン四酢酸二ナトリウム塩（EDTA-2ナトリウム）を1.6g。 、のdH 2 O 500mlに溶解し、pHを6.5に調整し、0.22 mmのフィルターを介して精製した溶液を濾過します。 </li><li> PBEバッファー20ml中のL-システインの0.035グラムを溶解します。溶液および滅菌パパイン酵素を100μlをフィルタリングします。 </li></ol></li><li>準備ができてアッセイを実施する場合には、-20°Cからチューブを取り、凍結されたヒドロゲルに準備パパイン溶液を300μlを添加します。乳鉢と乳棒を用いてゲルを粉砕し、モータ乳棒でよく混ぜます。 </li><li>追加のパパイン溶液で500μlにボリュームを持参してください。対照ゲルと同じ手順を実行します。 16時間3,9、60℃でインキュベートします。消化されたヒドロゲルを含む溶液は、従って、DNAおよびGAGの定量のために使用することができます。標準以下の製造業者のPRとしてラムダファージDNAとのピコグリーンアッセイを用いてDNA含量を測定otocol。 </li><li> 1,9-ジメチルメチレンブルー（DMMB）標準10としてサメのコンドロイチン硫酸と色素結合アッセイを使用して硫酸化GAG含有量を定量化します。対応湿重量のために、GAGの量を分割することにより、GAGの内容を決定します。 </li></ol> 6.機械的試験<ol><li> 10 Nロードセルを取り付けたインストロン5944試験システムを用いて、一軸圧縮試験を行います。 <ol><li> インビトロ培養の希望日後、細胞ヒドロゲル足場および無細胞制御ハイドロゲルの圧縮試験を行います。 </li><li> RTでPBS浴中で標本を水没し、負荷条件の下で軟骨組織が ​​経験する生理学的な歪みは10〜20％であると報告されているので、15％の最大ひずみ11,12に1％のひずみ/秒の速度で圧縮します13,14。 </li></ol></li><li>三次多項式を用いたひずみ曲線と曲線フィット対ストレスを作成します。 COMPRを決定15％の歪み値でカーブフィット式から能格接線係数。 </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.

このプロトコールで報告されたように、3Dヒドロゲルは、通常、単層培養物15で遭遇する線維軟骨細胞への細胞脱分化の過程を回避し、培養中の軟骨細胞表現型を維持することができます。また、chondrocytes-ヒドロゲル構築物の長期培養は、年齢や疾患に関連する本質的な細胞機能を維持し、良好な環境を明らかにしました。 3次元生体模倣ヒドロゲルの使用は、いくつかの利点を有します。まず、コンドロイチン硫酸（CS）、関節軟骨に見出される主要なコンポーネントを含めることは、コンドロイチナーゼを分泌することによってヒドロゲルマトリックスを分解し、新たに合成された軟骨細胞外マトリックス5、16を敷設するために細胞を可能にします。また、CSが示されています関節炎の関節における抗炎症特性を有すること。生体模倣ヒドロゲルはまた、軟骨修復における細胞送達のための足場材料として用いることができ、化学的に修飾することができますより良い組織生体材料統合17,18を容易にします 。 PEG-CSのヒドロゲルの使用は、長期的な軟骨細胞の文化や生化学的および機械的特性を評価することができます。ここでは、このプラットフォームは、軟骨工学のための最適な細胞タイプを定義するために、分化した軟骨細胞のさまざまなソースの比較分析のために有用であることができる方法を示しています。興味深いことに、ヒドロゲルにカプセル化された軟骨細胞は、それらの固有の能力に応じて生存したままと増殖します。 図2に示すように、ヒドロゲル組成物は、実際には、健康な若年成人軟骨細胞の増殖を支持する。グリコサミノグリカン（GAGによって評価機能細胞外マトリックスの沈着によって示されるように記述さヒドロゲルの組成及び構造はまた、軟骨組織の形成を促進します）定量。 追加の利点は、軟骨細胞ハイドロゲル構造物であります新たに形成された軟骨組織の機械的特性について評価することができます。一軸圧縮試験は、比較のために無細胞ヒドロゲル上で実行されるべきであることに注意してください。ヒドロゲルは、実際には、CSによる部分の剛性に固有の剛性を有します。負荷条件下で軟骨組織が ​​経験する生理学的な歪みが10~20であることが報告されているので（歪み率1％/ sで）5〜20％の一軸圧縮歪みが軟骨組織11,12の機械的試験に適用することができます％13,14。機械的試験への細胞を含んだし、無細胞ヒドロゲルの両方の応答は、培養エンドポイントで評価しました。私たちは、OA軟骨細胞を含む構築物の低い剛性とは対照的に、成人および若年性の軟骨細胞を含む構築物の同等の剛性を観察し、上記の例では。細胞 - ヒドロゲル構造体の機械的特性は、機能的特性の評価を可能にします細胞成熟性の詳細な分析を与えるに形成された組織。 結論として、軟骨組織を生成するために、別の軟骨細胞集団の可能性を研究するための3次元生体模倣ヒドロゲルの使用は、広く適用することができます。ここで説明したin vitro試験のほかに、細胞を含んだ構成物のin vivoでの移植は生理的な文脈で細胞成熟と再生可能性を研究するために想定することができます。追加の生体模倣因子とヒドロゲルプラットフォームのさらなる修飾はまた、軟骨細胞の増殖および成熟を最適化するように想定することができます。

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><tbody><tr><td>juvenile chondrocytes (Clonetics Normal Human Chondrocyte Cell System )</td><td>Lonza</td><td>CC-2550</td><td></td></tr><tr><td>adult chondrocytes (Clonetics 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&amp;nbsp;</td><td>2959</td><td></td></tr><tr><td>sodium chloride&amp;nbsp;</td><td>Sigma</td><td>S9888</td><td></td></tr><tr><td>chondroitin sulfate sodium salt&amp;nbsp;</td><td>Sigma</td><td>C9819</td><td></td></tr><tr><td>N-hydroxysuccinimide&amp;nbsp;</td><td>Sigma</td><td>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>516155</td><td></td></tr><tr><td>dialysis tubing</td><td>Spectrum Laboratories&amp;nbsp;</td><td>132700</td><td></td></tr><tr><td>Collagenase 2</td><td>Worthington Biochemical&amp;nbsp;</td><td>LS004177</td><td></td></tr><tr><td>Collagenase 4</td><td>Worthington Biochemical&amp;nbsp;</td><td>LS004189</td><td></td></tr><tr><td>DMEM/F12 media</td><td>HyClone, Thermo Scientific&amp;nbsp;</td><td>SH3002301&amp;nbsp;</td><td></td></tr><tr><td>live/dead assay</td><td>Life Technologies</td><td>&lt;a href=&quot;https://www.lifetechnologies.com/order/catalog/product/L3224&quot;&gt;L3224&lt;/a&gt;</td><td></td></tr><tr><td>Tri reagent</td><td>Life Technologies</td><td>AM9738</td><td></td></tr><tr><td>Quant-iT PicoGreen dsDNA Assay Kit</td><td>Invitrogen</td><td>&lt;a href=&quot;https://www.lifetechnologies.com/order/catalog/product/P11496&quot;&gt;P11496&lt;/a&gt;</td><td></td></tr><tr><td>Sodium phosphate dibasic&amp;nbsp;</td><td>Sigma</td><td>S3264</td><td></td></tr><tr><td>Ethylenediaminetetraacetic acid disodium salt&amp;nbsp;</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&amp;nbsp;</td><td>Sigma</td><td>341088</td><td></td></tr><tr><td>Instruments</td><td></td><td></td><td></td></tr><tr><td>UV light equipment - XX-15LW Bench Lamp, 365 nm</td><td>UVP</td><td>95-0042-07</td><td></td></tr><tr><td>Instron 5944 testing system&amp;nbsp;</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およびCS部分を含有する生理活性ヒドロゲル（ 図1）は、ヒト関節軟骨細胞2,3,5,7の文化と成熟のための理想的なプラットフォームを表します。異なる年齢および疾患状態の軟骨細胞は記載された方法を用いて培養し、それらの表現型、遺伝子発現、生成、軟骨組織の生化学的および機械的特性のために分析することができます。三軟骨細胞サンプル、若年6ヶ月、adult- 34年と74年osteoarthritic-は、3Dバイオミメティックハイドロゲルで培養3週間後に回収しました。遺伝子発現は、正常な軟骨細胞の分析、若年および成人の両方が、軟骨細胞遺伝子COL2A1およびCOL6A1の発現の増加を示しました。 COL6A1（ 図2）の発現を維持し、良好なバイオミメティック環境で培養されているにもかかわらず、軟骨形成表現型の損失を示しながら逆に、病気の軟骨細胞は、COL2A1の劇的な減少を示しました。 CS-PEGヒドロゲルはまた、軟骨細胞、増殖PEGおよびCSの既存のマトリックスを消化し、その細胞周囲および細胞外マトリックスを堆積させる、成熟した軟骨組織の主要な構成要素のための動的な環境として働きます。これらの能力を考えると、 インビトロ培養の3週間後の軟骨細胞の拡大は、ピコグリーン色素とDNAを定量することによって推定することができます。細胞の三つのグループの比較分析は、OA軟骨細胞は、培養の1日目に比べて劇的な減少を示しながら、少年と成人の集団の細胞密度が変化しなかったことを示しています。 DNA含量の損失はまた、長期培養（ 図3）の間の可能な細胞死を示唆している他のOA軟骨細胞のためではなく、観察されました。分泌されたマトリックスは、培養の3週間後に終点段階でDMMB色素結合アッセイにより硫酸化GAG含有量として定量化しました。 GAG含量はrecorとしてヒドロゲルの湿重量によって正規化されますDED酵素消化および無細胞の貢献の前に減算されます。 図4に示すように、軟骨は、3Dヒドロゲルにおける培養の3週間GAGの同様の有意な量の堆積します。 物理的な足場の存在は、一軸圧縮試験の2,3を通じてサンプルの生体力学的特性を評価することができます。無細胞ヒドロゲルは、圧縮弾性率の低下を受けている間、細胞を含んだ構築物は、培養3週間後（ 図4）の後に、圧縮弾性率を維持しました。表現型は、ここで説明する生化学的および生体力学的分析は、エンジニアリング軟骨のための異なった軟骨細胞集団の可能性を評価し、理解するのに有用です。 <img alt="図1" src="/files/ftp_upload/53085/53085fig1.jpg" /> 図3Dの軟骨細胞CUL 1. PEG-CS生体模倣ヒドロゲルチャー。軟骨細胞は、ポリ（エチレングリコールジアクリレート）（PEGDA）およびコンドロイチン硫酸-メタクリレート（CS-MA）を含む混合物中に再懸濁し、カスタムメイドの円筒ゲルモールド内に鋳造されています。 UV露光した後、ゲルを型から収集され、細胞の生存率がライブ死んで染色後24時間カプセル化することによって評価される固化。 <a href="https://www.jove.com/files/ftp_upload/53085/53085fig1large.jpg" target="_blank">この図の拡大版をご覧になるにはこちらをクリックしてください。</a> <img alt="図2" src="/files/ftp_upload/53085/53085fig2.jpg" /> 3Dバイオミメティックハイドロゲルで培養したヒト軟骨細胞における軟骨形成の遺伝子の発現を図2。少年、成人およびOA軟骨細胞による軟骨マーカーCOL2A1とCOL6A1の定量的遺伝子発現3Dバイオミメティックハイドロゲルで培養3週間後。値は、1日目誤差Bでの遺伝子の発現レベルに正規化されていますARSは、平均±SDを表します。 P、両側スチューデントt検定によって決定される* &lt;0.05。 Smeriglio らから変更された。2 <a href="https://www.jove.com/files/ftp_upload/53085/53085fig2large.jpg" target="_blank">この図の拡大版をご覧になるにはこちらをクリックしてください。</a> <img alt="図3" src="/files/ftp_upload/53085/53085fig3.jpg" /> 3Dバイオミメティックハイドロゲルで培養したヒト軟骨細胞における図3のDNA含有量。3Dバイオミメティックハイドロゲルで培養3週間後、少年、成人およびOA軟骨細胞におけるピコグリーンアッセイによるDNA定量。値は、1日目にDNAレベルに正規化されている<a href="https://www.jove.com/files/ftp_upload/53085/53085fig3large.jpg" target="_blank">。この図の拡大版をご覧になるにはこちらをクリックしてください。</a> <img alt="図4" src="/files/ftp_upload/53085/53085fig4.jpg" /> &lt;BR /&gt; 図4のGAG含有量測定と1日目と3次元バイオミメティックハイドロゲルで培養3週間後のヒト軟骨細胞の一軸圧縮試験を行った。1日目とで培養3週間後の軟骨細胞におけるDMMBアッセイによって（A）のGAG定量3Dバイオミメティックハイドロゲル。値は重量（WW）を濡らすように正規化され、μgの/ gと表現した。1日目と3次元バイオミメティックハイドロゲルで培養3週間後の無細胞と細胞を含んだヒドロゲルの（B）圧縮弾性率（kPaで）。 <a href="https://www.jove.com/files/ftp_upload/53085/53085fig4large.jpg" target="_blank">ご覧になるにはこちらをクリックしてくださいこの図の拡大版。</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.

関節軟骨細胞培養軟骨の生成のための3Dハイドロゲルの足場

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

20.0K ビュー.  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. 

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

Preparation of Chitosan-based Injectable Hydrogels and Its Application in 3D Cell Culture

The protocol presents a facile, efficient, and versatile method to prepare chitosan-based hydrogels using dynamic imine chemistry. The hydrogel is prepared by mixing solutions of glycol chitosan with a synthesized benzaldehyde terminated polymer gelator, and hydrogels are efficiently obtained in several minutes at room temperature. By varying ratios between glycol chitosan, polymer gelator, and water contents, versatile hydrogels with different gelation times and stiffness are obtained. When damaged, the hydrogel can recover its appearances and modulus, due to the reversibility of the dynamic imine bonds as crosslinkages. This self-healable property enables the hydrogel to be injectable since it can be self-healed from squeezed pieces to an integral bulk hydrogel after the injection process. The hydrogel is also multi-responsive to many bio-active stimuli due to different equilibration statuses of the dynamic imine bonds. This hydrogel was confirmed as bio-compatible, and L929 mouse fibroblast cells were embedded following standard procedures and the cell proliferation was easily assessed by a 3D cell cultivation process. The hydrogel can offer an adjustable platform for different research where a physiological mimic of a 3D environment for cells is profited. Along with its multi-responsive, self-healable, and injectable properties, the hydrogels can potentially be applied as multiple carriers for drugs and cells in future bio-medical applications.

Here we describe a facile preparation of chitosan-based injectable hydrogels using dynamic imine chemistry. Methods to adjust the hydrogel&#8217;s mechanical strength and its application in 3D cell culture are presented.

キトサンを用いた注射ゲルおよび 3 D 細胞培養への応用の準備

The protocol presents a facile, efficient, and versatile method to prepare chitosan-based hydrogels using dynamic imine chemistry. The hydrogel is ...

化学

Human Cartilage Tissue Fabrication Using Three-dimensional Inkjet Printing Technology

Bioprinting, which is based on thermal inkjet printing, is one of the most attractive enabling technologies in the field of tissue engineering and regenerative medicine. With digital control&#160;cells, scaffolds, and growth factors can be precisely deposited to the desired two-dimensional (2D) and three-dimensional (3D) locations rapidly. Therefore, this technology is an ideal approach to fabricate tissues mimicking their native anatomic structures. In order to engineer cartilage with native zonal organization, extracellular matrix composition (ECM), and mechanical properties, we developed a bioprinting platform using a commercial inkjet printer with simultaneous photopolymerization capable for 3D cartilage tissue engineering. Human chondrocytes suspended in poly(ethylene glycol) diacrylate (PEGDA) were printed for 3D neocartilage construction via layer-by-layer assembly. The printed cells were fixed at their original deposited positions, supported by the surrounding scaffold in simultaneous photopolymerization. The mechanical properties of the printed tissue were similar to the native cartilage. Compared to conventional tissue fabrication, which requires longer UV exposure, the viability of the printed cells with simultaneous photopolymerization was significantly higher. Printed neocartilage demonstrated excellent glycosaminoglycan (GAG) and collagen type II production, which was consistent with gene expression. Therefore, this platform is ideal for accurate cell distribution and arrangement for anatomic tissue engineering.

The methods described in this paper show how to convert a commercial inkjet printer into a bioprinter with simultaneous UV polymerization. The printer is capable of constructing 3D tissue structure with cells and biomaterials. The study demonstrated here constructed a 3D neocartilage.

三次元インクジェット印刷技術を用いて、ヒト軟骨組織の作製

Bioprinting, which is based on thermal inkjet printing, is one of the most attractive enabling technologies in the field of tissue engineering and ...

生物工学

Layered Alginate Constructs: A Platform for Co-culture of Heterogeneous Cell Populations

Many load bearing tissues possess structurally and functionally distinct regions, typically accompanied by different cell phenotypes with differential mechanosensing characteristics. Engineering and analysis of these tissue types remain a challenge. Layered hydrogel constructs provide an opportunity for investigating the interactions among multiple cell populations within single constructs. Alginate hydrogels are both biocompatible and allow for easy isolation of cells after experimentation. Here, we describe a method for the development of small sized dual layered alginate hydrogel discs. This process maintains high cell viability of human mesenchymal stem cells during the formation process and these layered discs can withstand unconfined cyclic compression, commonly used for stimulation of hMSCs undergoing chondrogenesis. These layered constructs can potentially be scaled up to include additional levels, and also be used to segregate cell populations initially after layering. This dual layer alginate hydrogel culture platform can be used for many different applications including engineering and analysis of cells of load bearing tissues and co-cultures of other cell types.

Engineering and analysis of load bearing tissues with heterogeneous cell populations are still a challenge. Here, we describe a method for creating bi-layered alginate hydrogel discs as a platform for co-culture of diverse cell populations within one construct.

階層化アルギン酸構築：異種細胞集団の共培養のためのプラットフォーム

Many load bearing tissues possess structurally and functionally distinct regions, typically accompanied by different cell phenotypes with differential ...

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.

軟骨再生のための3次元磁気幹細胞凝集およびバイオリアクター成熟

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

Culturing Mammalian Cells in Three-dimensional Peptide Scaffolds

A useful technique for culturing cells in a self-assembling nanofiber three-dimensional (3D) scaffold is described. This culture system recreates an environment that closely mimics the structural features of non-polarized tissue. Furthermore, the particular intrinsic nanofiber structure of the scaffold makes it transparent to visual light, which allows for easy visualization of the sample under microscopy. This advantage was largely used to study cell migration, organization, proliferation, and differentiation and thus any development of their particular cellular function by staining with specific dyes or probes. Furthermore, in this work, we describe the good performance of this system to easily study the redifferentiation of expanded human articular chondrocytes into cartilaginous tissue. Cells were encapsulated into self-assembling peptide scaffolds and cultured under specific conditions to promote chondrogenesis. Three-dimensional cultures showed good viability during the 4 weeks of the experiment. As expected, samples cultured with chondrogenic inducers (compared to non-induced controls) stained strongly positive for toluidine blue (which stains glycosaminoglycans (GAGs) that are highly present in cartilage extracellular matrix) and expressed specific molecular markers, including collagen type I, II and X, according to Western Blot analysis. This protocol is easy to perform and can be used at research laboratories, industries and for educational purposes in laboratory courses.

Here, we present a protocol to obtain 3D-culture systems in self-assembling peptide scaffolds to promote the differentiation of dedifferentiated human articular chondrocytes into cartilage-like tissue.

三次元ペプチド足場で哺乳動物の細胞を培養

A useful technique for culturing cells in a self-assembling nanofiber three-dimensional (3D) scaffold is described. This culture system recreates an ...

A 3D System for Culturing Human Articular Chondrocytes in Synovial Fluid

Cartilage destruction is a central pathological feature of osteoarthritis, a leading cause of disability in the US. Cartilage in the adult does not regenerate very efficiently in vivo; and as a result, osteoarthritis leads to irreversible cartilage loss and is accompanied by chronic pain and immobility 1,2. Cartilage tissue engineering offers promising potential to regenerate and restore tissue function. This technology typically involves seeding chondrocytes into natural or synthetic scaffolds and culturing the resulting 3D construct in a balanced medium over a period of time with a goal of engineering a biochemically and biomechanically mature tissue that can be transplanted into a defect site in vivo 3-6. Achieving an optimal condition for chondrocyte growth and matrix deposition is essential for the success of cartilage tissue engineering. 
 In the native joint cavity, cartilage at the articular surface of the bone is bathed in synovial fluid. This clear and viscous fluid provides nutrients to the avascular articular cartilage and contains growth factors, cytokines and enzymes that are important for chondrocyte metabolism 7,8. Furthermore, synovial fluid facilitates low-friction movement between cartilaginous surfaces mainly through secreting two key components, hyaluronan and lubricin 9 10. In contrast, tissue engineered cartilage is most often cultured in artificial media. While these media are likely able to provide more defined conditions for studying chondrocyte metabolism, synovial fluid most accurately reflects the natural environment of which articular chondrocytes reside in.
 Indeed, synovial fluid has the advantage of being easy to obtain and store, and can often be regularly replenished by the body. Several groups have supplemented the culture medium with synovial fluid in growing human, bovine, rabbit and dog chondrocytes, but mostly used only low levels of synovial fluid (below 20&#37;) 11-25. While chicken, horse and human chondrocytes have been cultured in the medium with higher percentage of synovial fluid, these culture systems were two-dimensional 26-28. Here we present our method of culturing human articular chondrocytes in a 3D system with a high percentage of synovial fluid (up to 100&#37;) over a period of 21 days. In doing so, we overcame a major hurdle presented by the high viscosity of the synovial fluid. This system provides the possibility of studying human chondrocytes in synovial fluid in a 3D setting, which can be further combined with two other important factors (oxygen tension and mechanical loading) 29,30 that constitute the natural environment for cartilage to mimic the natural milieu for cartilage growth. Furthermore, This system may also be used for assaying synovial fluid activity on chondrocytes and provide a platform for developing cartilage regeneration technologies and therapeutic options for arthritis.

A 3D system of culturing human articular chondrocytes in high levels of synovial fluid is described. Synovial fluid reflects the most natural microenvironment for articular cartilage, and can be easily obtained and stored. This system thus can be used for studying cartilage regeneration and for screening therapeutics for treating arthritis.

滑液における培養ヒト関節軟骨細胞用の3Dシステム

Cartilage destruction is a central pathological feature of osteoarthritis, a leading cause of disability in the US. Cartilage in the adult does not ...

生物学

Mechanical Stimulation of Chondrocyte-agarose Hydrogels

Articular cartilage suffers from a limited repair capacity when damaged by mechanical insult or degraded by disease, such as osteoarthritis. To remedy this deficiency, several medical interventions have been developed. One such method is to resurface the damaged area with tissue-engineered cartilage; however, the engineered tissue typically lacks the biochemical properties and durability of native cartilage, questioning its long-term survivability. This limits the application of cartilage tissue engineering to the repair of small focal defects, relying on the surrounding tissue to protect the implanted material. To improve the properties of the developed tissue, mechanical stimulation is a popular method utilized to enhance the synthesis of cartilaginous extracellular matrix as well as the resultant mechanical properties of the engineered tissue. Mechanical stimulation applies forces to the tissue constructs analogous to those experienced in vivo. This is based on the premise that the mechanical environment, in part, regulates the development and maintenance of native tissue1,2. The most commonly applied form of mechanical stimulation in cartilage tissue engineering is dynamic compression at physiologic strains of approximately 5-20% at a frequency of 1 Hz1,3. Several studies have investigated the effects of dynamic compression and have shown it to have a positive effect on chondrocyte metabolism and biosynthesis, ultimately affecting the functional properties of the developed tissue4-8. In this paper, we illustrate the method to mechanically stimulate chondrocyte-agarose hydrogel constructs under dynamic compression and analyze changes in biosynthesis through biochemical and radioisotope assays. This method can also be readily modified to assess any potentially induced changes in cellular response as a result of mechanical stimuli.

The biosynthesis of cartilaginous extracellular matrix by chondrocytes can be affected by application of mechanical stimuli. This method describes the technique of applying dynamic compressive strains to chondrocytes encapsulated in 3D constructs and the evaluation of induced changes in chondrocyte metabolism.

軟骨アガロースゲルの機械的刺激

Articular cartilage suffers from a limited repair capacity when damaged by mechanical insult or degraded by disease, such as osteoarthritis. To remedy ...

Creation of a Knee Joint-on-a-Chip for Modeling Joint Diseases and Testing Drugs

The high prevalence of debilitating joint diseases like osteoarthritis (OA) poses a high socioeconomic burden. Currently, the available drugs that target joint disorders are mostly palliative. The unmet need for effective disease-modifying OA drugs (DMOADs) has been primarily caused by the absence of appropriate models for studying the disease mechanisms and testing potential DMOADs. Herein, we describe the establishment of a miniature synovial joint-mimicking microphysiological system (miniJoint) comprising adipose, fibrous, and osteochondral tissue components derived from human mesenchymal stem cells (MSCs). To obtain the three-dimensional (3D) microtissues, MSCs were encapsulated in photocrosslinkable methacrylated gelatin before or following differentiation. The cell-laden tissue constructs were then integrated into a 3D-printed bioreactor, forming the miniJoint. Separate flows of osteogenic, fibrogenic, and adipogenic media were introduced to maintain the respective tissue phenotypes. A commonly shared stream was perfused through the cartilage, synovial, and adipose tissues to enable tissue crosstalk. This flow pattern allows the induction of perturbations in one or more of the tissue components for mechanistic studies. Furthermore, potential DMOADs can be tested via either &#34;systemic administration&#34; through all the medium streams or &#34;intraarticular administration&#34; by adding the drugs to only the shared &#34;synovial fluid&#34;-simulating flow. Thus, the miniJoint can serve as a versatile in vitro platform for efficiently studying disease mechanisms and testing drugs in personalized medicine.

We provide detailed methods for generating four types of tissues from human mesenchymal stem cells, which are used to recapitulate the cartilage, bone, fat pad, and synovium in the human knee joint. These four tissues are integrated into a customized bioreactor and connected through microfluidics, thus generating a knee joint-on-a-chip.

関節疾患のモデリングと薬剤検査のための膝関節チップの作成

The high prevalence of debilitating joint diseases like osteoarthritis (OA) poses a high socioeconomic burden. Currently, the available drugs that target ...

Synthesis of Decellularized Cartilage Extracellular Matrix Hydrogels

Decellularized cartilage extracellular matrix (DC-ECM) hydrogels are promising biomaterials for tissue engineering and regenerative medicine due to their biocompatibility and ability to mimic natural tissue properties. This protocol aims to produce DC-ECM hydrogels that closely mimic the native ECM of cartilage tissue. The protocol involves a combination of physical and chemical disruption and enzymatic digestion to remove the cellular material while preserving the structure and composition of the ECM. The DC-ECM is cross-linked using a chemical agent to form a stable and biologically active hydrogel. The DC-ECM hydrogel has excellent biological activity, spatial structure, and biological induction function, as well as low immunogenicity. These characteristics are beneficial in promoting cell adhesion, proliferation, differentiation, and migration and for creating a superior microenvironment for cell growth. This protocol provides a valuable resource for researchers and clinicians in the field of tissue engineering. Biomimetic hydrogels can potentially enhance the development of effective tissue engineering strategies for cartilage repair and regeneration.

This paper introduces a new method for the synthesis of decellularized cartilage extracellular matrix (DC-ECM) hydrogels. DC-ECM hydrogels have excellent biocompatibility and provide a superior microenvironment for cell growth. Therefore, they can be ideal cell scaffolds and biological delivery systems.

脱細胞化軟骨細胞外マトリックスハイドロゲルの合成

Decellularized cartilage extracellular matrix (DC-ECM) hydrogels are promising biomaterials for tissue engineering and regenerative medicine due to their ...

Generating Spheroids from Various Chondrocytes using Low-Adhesive Conditions under Gravity and Homemade Mini-Bioreactors

Cartilage repair in chronic joint diseases demands advanced cell-based therapies to regenerate damaged tissues effectively. This protocol provides a step-by-step method for differentiating induced pluripotent stem cells (iPSCs) into chondrocyte-based spheroids, supporting tissue engineering and cell therapy applications. The differentiation process is carefully structured to promote chondrogenic lineage commitment, beginning with iPSCs cultured in specific media that sequentially guide cells through critical stages of differentiation. Initially, iPSCs are expanded to reach optimal confluency before induction toward chondrogenic lineage using a series of defined media changes. By day 10, cells are transitioned to a chondrogenesis-promoting medium that enhances the formation of chondrocyte-like cells expressing key markers of mature chondrocytes. Further aggregation in 96-well agarose-coated plates leads to the formation of three-dimensional spheroids, which are then cultured in custom mini-bioreactors designed to simulate a microenvironment that encourages extracellular matrix (ECM) deposition. By enabling scalable production of chondrocyte spheroids that mimic native cartilage characteristics, this approach offers a promising, reproducible solution for developing cell-based treatments for cartilage defects, providing broad utility for clinical and research applications in musculoskeletal regenerative medicine.

This study describes a method for producing chondrocytic spheroids by aggregating cells into spheroids under low-adhesion conditions using gravity, followed by culturing the resulting spheroids in mini-bioreactors.

重力下での低接着条件と自家製ミニバイオリアクターを用いた種々の軟骨細胞からのスフェロイド作製

Cartilage repair in chronic joint diseases demands advanced cell-based therapies to regenerate damaged tissues effectively. This protocol provides a ...