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 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.

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

19.8K 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

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.

一个简单的悬滴细胞生成三维球体文化协议

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

科研

生物工程

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.

制备纤维蛋白三维支架干细胞培养应用

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.

自我报告的支架3维细胞培养

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.

用脱细胞支架龙ESC衍生鼠气道上皮细胞的生成

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.

实验和有限元议定书调查中性和带电溶质的跨关节软骨运输

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

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

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

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

3D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 ...

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.

Bioink Precellularization 无源混合装置打印软骨及皮肤组织类似物的应用

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.

全完整型小鼠软骨外种机械负荷对软骨细胞损伤的实时可视化与分析

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.

脱碳软骨衍生基质支架的制备

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.

三维印刷多孔纤维素纳米复合水凝胶脚手架

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