Name: 3d hydrogel scaffolds for articular chondrocyte culture and cartilage generation
Uploaded: 2015-10-07T14:00:00.000+00:00
Duration: 12 min 37 s
Description: Watch this Scientific Journal Video about 3D Hydrogel Scaffolds for Articular Chondrocyte Culture and Cartilage Generation at JoVE.com

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.

所有实验均按照斯坦福大学人类受试者的指南进行批准机构审查委员会的协议。 1.关节软骨细胞分离<ol><li>从组织全膝关节置换术中放弃获取软骨和剖析如前面8所述。 </li><li>视觉上选择软骨样本用于光滑表面的内侧或外侧股骨髁，使切口在软骨的表面通过手术刀以除去4-5毫米软骨活检而不影​​响软骨下骨。 </li><li>通过用O / N的治疗用纯化的胶原酶的组织的酶解隔离来自细胞外基质的软骨细胞。使用方法1:在软骨细胞生长培养基1比胶原酶II（125 U / ml）和胶原酶IV（160单位/毫升）在37℃。 </li><li>翌日，过滤消化的组织通过一个70包含游离细胞毫米孔径的尼龙网。用20ml的DMEM / F12和离心机在600×g离心5分钟，洗两次。 </li><li>板分离的软骨细胞以1×10 6个细胞/ 60毫米的培养皿的密度以下重新悬浮在DMEM / F12中补充有10％胎牛血清（FBS），25微克/毫升抗坏血酸，2mM的L-谷氨酰胺，抗菌剂（100U / ml青霉素，100微克/毫升链霉素和0.25微克/毫升两性霉素B）。保持所述板在37℃和维持初级软骨细胞培养物作为高密度的单层封装前在仿生水凝胶。 </li></ol> 2.仿生水凝胶的制备注意:此方法允许将包含聚（乙二醇二丙烯酸酯）（PEGDA，MW 5,000个克/摩尔），硫酸软骨素 - 甲基丙烯酸酯（CS-MA）的DPBS仿生水凝胶的合成。加入光引发剂使光敏交联。最终水凝胶组合物中含有7％的重量吨/体积（w / v）的PEGDA的，3％w / v的CS-MA的，和0.05％w / v的光引发剂。 <ol><li>合成CS-MA由政务司司长官能聚合物与甲基丙烯酸酯基。 <ol><li>通过将1.952克的MES和5.84克氯化钠在200ml的去离子水（DIH 2 O）的制备为50mM 2-吗啉乙磺酸（MES）和0.5M氯化钠（NaCl）中缓冲的溶液。溶解5克硫酸软骨素钠盐在缓冲溶液中。 </li><li>加0.532克（4.62毫摩尔）N-羟基琥珀酰亚胺（NHS）和1.771克（9.24毫摩尔）1-乙基-3-（3-二甲氨基丙基） - 碳二亚胺盐酸盐（EDC）（NHS的摩尔比:EDC = 1:2），以该溶液中，搅拌5分钟。 </li><li>加0.765克（4.62毫摩尔）2-氨基乙基甲基丙烯酸酯（AEMA）（NHS的摩尔比:EDC:AEMA = 1:2:1），以该溶液并保持在RT 24小时，使反应。 </li><li>净化透析对去离子水（DIH 2 O）的混合物4天使用透析管（12-14 kDa的截留分子量）。 </li><li>过滤器在p通过0.22微米的过滤器urified溶液并放置含有在干燥器和真空O / N的溶液开口管中。确保所有管避光。存储所述溶解的聚合物在-20℃下从光和水分的保护。 </li></ol></li></ol> 3.电池封装<ol><li>高压釜在PCR膜和圆柱形杆（用于冲压出细胞载货三维水凝胶），并通过浸没在0.2微米的过滤在组织培养罩70％的乙醇在UV光下消毒定做圆柱形凝胶铸型。 </li><li>密封凝胶铸型用高压灭菌的PCR膜的底部没有气泡或缝隙，以防止泄漏。保持它在无菌150毫米板。 </li><li>之前在仿生水凝胶电池封装一天，分离高密度的软骨细胞单层与O / N治疗胶原酶Ⅱ和Ⅳ型胶原酶的解决方案。用125 U / ml的和胶原酶II 160 U / ml的胶原酶VI每个软骨细胞生长培养基在37℃。 </li><li>取50毫升的管中，添加5％重量/体积（w / v）的聚（乙二醇二丙烯酸酯）（PEGDA，MW = 5,000克/摩尔），3％w / v的硫酸软骨素 - 甲基丙烯酸酯（CS-MA）和0.05％瓦特/ DPBS v光引发剂。涡管混合溶液中，并保持它放在一边。由轻保护管。 </li><li>收集解离的细胞在50ml Falcon管中，在460×g离心计数，离心5分钟。抽吸所有从细胞管的介质，并添加上述混合凝胶材料以重新悬浮细胞以15×10 6个细胞/ ml的密度。混合它30倍彻底，同时避免气泡的形成。 </li><li>吸移管72微升细胞悬浮液的水凝胶或水凝胶单独进定做圆柱形凝胶模具和诱导通过暴露于UV光（波长365nm）的凝胶化在3毫瓦/米2 5分钟。准备没有单元格内容的一些水凝胶的解决方案。使用这些结构作为阴性对照未来的生化和机械测试。 Measu重的UV光的UV强度计的强度。以调整强度，改变UV光源和凝胶化过程中，水凝胶的支架之间的距离。 注:由于水凝胶含有CS，无细胞贡献从细胞载货水凝胶的生化GAG含量减去以仅代表蜂窝GAG。 </li><li>用手术刀切膜容易剥离，并小心地将其删除。与圆柱形棒的帮助下，压出凝胶成6孔板用5ml无菌的DPBS洗掉未聚合凝胶和松散的细胞。 </li><li>培养在含有1.5 ml的软骨细胞生长培养基到每个孔24孔板的细胞载货水凝胶。使用一次性抹刀到洗涤水凝胶转移到井中。 </li><li>使用存活率/细胞毒试剂盒由活死染色后24小时封装评估细胞生存力。文化细胞载货凝胶和水凝胶的空3-6周更换新鲜的软骨细胞GRO相机连前每2天收获进行分析的媒体。 </li></ol> 4. RNA提取和基因表达分析<ol><li>小心吸媒体，并把无菌PBS 1X在细胞载货以及空控制水凝胶。 </li><li>用无菌刮铲的帮助下，各水凝胶转移到一个干净的1.5 ml微量离心管中并加入250微升三试剂的每个管。按照制造商的说明分离RNA。 </li><li> RNA分离和定量使用从每个样品1微克，并经过使用高容量的cDNA反转录试剂盒进行逆转录成cDNA。 </li><li>对于定量PCR荧光定量使用基因表达阵列检查你的目的基因的表达。内部正常化的基因表达水平对于GAPDH。 </li><li>用于PCR条件包括2分钟温育在50℃下灭活先前扩增子用尿嘧啶DNA糖基化酶，随后是1060;分钟温育在95℃下以激活Taq聚合酶。然后在95℃下进行PCR的40个循环，由15秒，并在60℃下1分钟。 </li></ol> 5.生化分析<ol><li>为每个小区构建的水凝胶量化DNA和硫酸化糖胺聚糖（sGAG）生产如下。 </li><li>小心吸媒体，并把无菌PBS 1X在细胞载货以及空控制水凝胶。称量空管和纸巾吸干后把水凝胶内，并记录水凝胶的重量或湿重。 </li><li>冻结各水凝胶在-20℃在一个单独的1.5管酶消化毫升的微量，并随后将经消化的产物的等分试样运行PicoGreen检测结果（对于DNA）和（为GAG）二甲基亚甲蓝测定。 </li><li>准备水凝胶的酶消化的木瓜蛋​​白酶溶液。 <ol><li>首先称取7.1克钠phosphat的准备PBE缓冲区ë二元（ 磷酸氢二钠）和1.6克乙二胺四乙酸二钠盐（EDTA钠2）的。溶解在500毫升的dh 2 O为将pH调节至6.5，并过滤通过0.22毫米过滤纯化的溶液。 </li><li>溶解0.035克L-半胱氨酸的20毫升的PBE缓冲器。过滤溶液和100微升无菌木瓜蛋白酶的酶。 </li></ol></li><li>当准备好执行测定，取管为-20℃，并添加300微升准备木瓜蛋白酶溶液的冷冻凝胶。粉碎用研钵和研杵的凝胶，并与马达杵拌匀。 </li><li>使体积达到500微升用另外的木瓜蛋白酶溶液。为控制凝胶执行相同的过程。孵育在60℃下16小时3,9。因此可用于DNA和GAG定量含有消化的水凝胶溶液中。测量以下制造商的公关与使用λ噬菌体DNA作为标准的PicoGreen检测结果的DNA含量otocol。 </li><li>使用1,9-二甲基亚甲蓝（DMMB）与鲨鱼硫酸软骨素作为标准的10染料结合分析量化硫酸化GAG含量。除以GAG的量为相应的湿重确定GAG含量。 </li></ol> 6.机械测试<ol><li>执行使用配有10N的测力传感器的Instron 5944测试系统无侧限压缩试验。 <ol><li> 在体外培养的所需天之后，进行在细胞水凝胶支架和非细胞控制水凝胶的压缩试验。 </li><li>淹没在PBS浴中的试样在RT和在1％应变/秒的速率压缩至15％11,12的最大应变，因为生理应变由软骨组织荷条件下经历已报道为10-20％ 13,14。 </li></ol></li><li>创建应力与使用一个三阶多项式方程应变曲线和曲线拟合。确定COMPRessive切线模量从15％应变值的曲线拟合方程。 </li></ol>

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The authors have nothing to disclose.

正如在这个协议中，三维水凝胶是能够维持软骨细胞表型中培养，以避免细胞去分化的过程分成纤维软骨细胞通常与单层培养15遇到。此外，水凝胶chondrocytes-构建体的长期培养表明，保持与年龄和疾病相关的固有细胞特征的良好环境。 使用三维仿生水凝胶有几个优点。首先，将包含硫酸软骨素（CS），在关节软骨中的主要组成部分，使细胞通过分泌软骨素酶以降解水凝胶基质和放下新合成的软骨细胞外基质5，16，另外，CS已经显示具有抗发炎的特性，在关节炎关节。仿生水凝胶也可以用作支架材料用于软骨修复细胞递送，并且可以被化学修饰为更好地组织生物材料的集成17,18。 使用的PEG-CS凝胶的允许软骨细胞的长期培养和生物化学和机械性能的评价。这里，我们显示如何这个平台可以以确定最佳的细胞类型为软骨工程为分化软骨细胞的各种来源的比较分析是有用的。有趣的是，软骨细胞包封在水凝胶保持存活并增殖按照其固有的能力。水凝胶组合物载体，事实上，健康青少年和成人的软骨细胞的生长，如图2所描述的水凝胶的组成和结构也促进软骨组织形成由功能性细胞外基质由糖胺评估的沉积（GAG所指示）定量。 一个额外的优点是，软骨细胞的水凝胶的结构可以评估新形成的软骨组织的机械性能。注意，无侧限压缩测试应在无细胞的水凝胶进行比较来执行。水凝胶，实际上，有一种内在的刚度由于在CS部分的刚性。 5-20％无侧限压缩变形（应变速率1％/秒）可应用于软骨组织11,12由于生理应变由软骨组织负载条件下所经历的机械测试已报道在10-20 ％13,14。两种细胞载货和无细胞水凝胶力学性能试验的反应在文化终点进行了评价。在所描述的实施例以上，我们观察到含有相对于含有OA软骨细胞的构建体的低刚度成人和少年软骨细胞的构建体可比较的刚度。细胞 - 凝胶构建体，例如机械性能允许的功能性质的评估形成的组织给予了深入的分析，细胞成熟能力。 总之，利用三维仿生水凝胶的研究不同软骨细胞群的潜在生成软骨组织可以广泛应用。除了 ​​这里所描述的在体外研究中，在体内移植的细胞载货构建体可以设想来研究细胞的成熟和再生潜能的生理环境。水凝胶平台具有附加仿生因素的进一步的修改，也可设想，以优化软骨细胞增殖和成熟。

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.

含有PEG和CS部分（ 图1）生物活性水凝胶代表了文化的人关节软骨细胞2,3,5,7和成熟的理想平台。从不同年龄和疾病状态的软骨细胞可以培养所描述的方法和它们的表型，基因表达和产生的软骨组织的生物化学和机械性能进行分析。三软骨细胞样本，juvenile- 6个月，adult-34年和osteoarthritic- 74岁，在3D仿生水凝胶3周培养后收获。的正常软骨细胞，既幼年和成年基因表达分析，显示增加的软骨细胞的基因的Col2a1和COL6A1的表达。相反的，患病的软骨细胞表现出的Col2a1的显着降低，同时保持COL6A1（图2）的表达示出尽管是培养在一个有利的仿生环境软骨表型的损失。 在CS-PEG水凝胶也作为软骨细胞增殖的动态环境，消化PEG和CS的预先存在的基质和沉积其细胞外和细胞外基质，成熟软骨组织的主要成分。鉴于这些能力，3周的体外培养后，软骨细胞扩张可以通过DNA与的PicoGreen染料的定量估计。的三组细胞的对比分析表明，少年和成年人口的细胞密度不变，而OA软骨细胞表现出显着降低相比培养的第1天。还观察到用于OA中而非其他软骨细胞的长期培养物（ 图3）中建议可能的细胞死亡的DNA含量的损失。分泌的基质3个星期培养后定量为硫酸化的GAG含量DMMB染料结合测定在终点处的阶段。 GAG含量是由水凝胶作为录音功的湿重标准化DED酶消化和脱细胞贡献之前被扣除。 如图4，软骨细胞中在三维水凝胶3周的培养的沉积相似的显著量的GAG的。 一个物理支架的存在允许样品的生物力学特性的评价通过无侧限压缩试验2,3。而脱水凝胶经受的压缩模量的降低，细胞载货构建体维持3周培养（图4）之后的压缩模量。的表型，这里所描述的生物化学和生物力学分析，因此，用于评估和理解不同软骨细胞群的工程软骨的潜力是有用的。 <img alt="图1" src="/files/ftp_upload/53085/53085fig1.jpg" /> 图1. PEG-CS仿生水凝胶的3D软骨细胞的小路TURE。软骨细胞再悬浮于含有聚（二丙烯酸乙二醇酯）（PEGDA）和硫酸软骨素-甲基丙烯酸酯（CS-MA）和浇铸到所述定做圆柱形凝胶模具的混合物。紫外线照射后，固化凝胶从模具中收集细胞活力是由活死细胞染色24小时后封装评估。 <a href="https://www.jove.com/files/ftp_upload/53085/53085fig1large.jpg" target="_blank">请点击此处查看该图的放大版本。</a> <img alt="图2" src="/files/ftp_upload/53085/53085fig2.jpg" /> 图中的人软骨细胞的三维仿生水凝胶中培养软骨细胞的基因表达2。软骨标记的Col2a1和COL6A1定量基因表达的少年，成人和OA软骨细胞的三维仿生水凝胶3周文化之后。值都被归到基因表达水平在每日1次错误BARS代表平均值±SD。 P * &lt;0.05如通过双尾Student t检验确定。从Smeriglio 等修饰<a href="https://www.jove.com/files/ftp_upload/53085/53085fig2large.jpg" target="_blank">。2，请点击此处查看该图的放大版本。</a> <img alt="图3" src="/files/ftp_upload/53085/53085fig3.jpg" /> 在人软骨细胞在3D仿生水凝胶中培养图3. DNA含量，DNA定量分析在青少年，成人及OA软骨细胞PicoGreen检测结果的三维仿生水凝胶3周文化之后。值都被归到DNA水平在第1天<a href="https://www.jove.com/files/ftp_upload/53085/53085fig3large.jpg" target="_blank">，请点击此处查看该图的放大版本。</a> <img alt="图4" src="/files/ftp_upload/53085/53085fig4.jpg" /> &lt;登记/&gt; 图4的GAG含量测量和人软骨细胞在1天周和3周培养在三维仿生水凝胶后的无侧限压缩试验。（A）中的GAG通过DMMB测定软骨细胞中在1天，并在3周的培养之后，量化3D仿生水凝胶。值都被归为湿重（WW），并表示为微克/克的无细胞和细胞载货凝胶1天3周文化的三维仿生水凝胶后（B）压缩模量（千帕）。 <a href="https://www.jove.com/files/ftp_upload/53085/53085fig4large.jpg" target="_blank">请点击这里查看一个更大的版本这个数字。</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水凝胶支架的关节软骨细胞培养及软骨生成

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 次观看.  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. 

视频: 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.

糖注射水凝胶的制备及其在3D 细胞培养中的应用

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

科研

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.

人体软骨组织制作采用三维喷墨打印技术

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

生物工程

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.

分层海藻酸钠构造:一个异质细胞群的共培养平台

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.

3D磁干细胞聚集和生物反应器为成熟软骨再生

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.

三维肽支架中哺乳动物细胞的培养

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.

一个培养人关节软骨细胞的三维系统在滑液

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.

机械性刺激软骨细胞琼脂糖凝胶

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.

创建膝关节芯片，用于关节疾病建模和药物测试

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.

脱细胞软骨细胞外基质水凝胶的合成

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.

在重力和自制微型生物反应器下使用低粘附条件从各种软骨细胞生成球状体

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