3D水凝胶支架的关节软骨细胞培养及软骨生成
Summary
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.
Abstract
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.
Introduction
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.
Protocol
Representative Results
Discussion
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。
Divulgaciones
The authors have nothing to disclose.
Acknowledgements
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.
Materials
| juvenile chondrocytes (Clonetics™ Normal Human Chondrocyte Cell System ) | Lonza | CC-2550 | |
| adult chondrocytes (Clonetics™ Normal Human Chondrocyte Cell System) | Lonza | CC-2550 | |
| poly(ethylene glycol diacrylate) | Laysan Bio | ACRL-PEG-ACRL-1000-1g | |
| 2-morpholinoethanesulfonic acid | Sigma | M5287 | |
| photoinitiator | Irgacure | 2959 | |
| sodium chloride | Sigma | S9888 | |
| chondroitin sulfate sodium salt | Sigma | C9819 | |
| N-hydroxysuccinimide | Sigma | 130672 | |
| 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride | Sigma | E1769 | |
| 2-aminoethyl methacrylate | Sigma | 516155 | |
| dialysis tubing | Spectrum Laboratories | 132700 | |
| Collagenase 2 | Worthington Biochemical | LS004177 | |
| Collagenase 4 | Worthington Biochemical | LS004189 | |
| DMEM/F12 media | HyClone, Thermo Scientific | SH3002301 | |
| live/dead assay | Life Technologies | L3224 | |
| Tri reagent | Life Technologies | AM9738 | |
| Quant-iT™ PicoGreen® dsDNA Assay Kit | Invitrogen | P11496 | |
| Sodium phosphate dibasic | Sigma | S3264 | |
| Ethylenediaminetetraacetic acid disodium salt | Sigma | E5134 | |
| L-Cysteine | Sigma | C1276 | |
| 1,9-dimethylmethylene blue | Sigma | 341088 | |
| Instruments | |||
| UV light equipment – XX-15LW Bench Lamp, 365nm | UVP | 95-0042-07 | |
| Instron 5944 testing system | Instron Corporation | E5940 |
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