जोड़ उपास्थिकोशिका संस्कृति और उपास्थि पीढ़ी के लिए 3 डी हाइड्रोजेल Scaffolds
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
इस प्रोटोकॉल में सूचना दी, 3 डी हाइड्रोजेल आमतौर पर monolayer संस्कृतियों 15 के साथ सामना करना तंतुपास्थि कोशिकाओं में सेल dedifferentiation की प्रक्रिया से बचने, संस्कृति में उपास्थिकोशिका फेनोटाइप बनाए रखने में सक्षम हैं। इसके अलावा, chondrocytes- हाइड्रोजेल निर्माण के लिए लंबी अवधि संस्कृतियों उम्र और बीमारी के साथ जुड़े आंतरिक सेल सुविधाओं का कहना है कि एक अनुकूल वातावरण का पता चला।
एक 3 डी biomimetic हाइड्रोजेल के उपयोग के कई फायदे हैं। सबसे पहले, chondroitin सल्फेट का समावेश (सीएस), जोड़ कार्टिलेज में पाया जाने वाला एक प्रमुख घटक है, chondroitinase स्रावित द्वारा हाइड्रोजेल मैट्रिक्स नीचा और अतिरिक्त सेलुलर मैट्रिक्स 5, 16 नए संश्लेषित उपास्थि नीचे रखना करने के लिए कोशिकाओं को सक्रिय करें। इसके अलावा, सीएस दिखाया गया है गठिया के संयुक्त में विरोधी भड़काऊ गुण है। biomimetic हाइड्रोजेल भी उपास्थि की मरम्मत में सेल डिलीवरी के लिए एक मचान सामग्री के रूप में इस्तेमाल किया जा सकता है, और रासायनिक संशोधित किया जा सकता हैबेहतर ऊतक biomaterial एकीकरण 17,18 सुविधा के लिए।
खूंटी-सीएस हाइड्रोजेल के उपयोग के लिए लंबी अवधि के chondrocytes की संस्कृतियों और जैव रासायनिक और यांत्रिक गुणों के मूल्यांकन की अनुमति देता है। यहाँ हम इस मंच उपास्थि इंजीनियरिंग के लिए इष्टतम सेल प्रकार परिभाषित करने के क्रम में भेदभाव chondrocytes के विभिन्न स्रोतों का तुलनात्मक विश्लेषण के लिए उपयोगी हो सकता है कि कैसे दिखा। दिलचस्प बात यह है हाइड्रोजेल में समझाया chondrocytes अपने आंतरिक क्षमता के अनुसार व्यवहार्य बने हुए हैं और पैदा करना। हाइड्रोजेल रचना का समर्थन करता है, वास्तव में, स्वस्थ किशोर और वयस्क chondrocytes के विकास चित्रा 2 में दिखाया गया है। वर्णित हाइड्रोजेल की संरचना और संरचना भी उपास्थि ऊतक गठन को बढ़ावा ग्लाइकोसमिनोग्लाइकन द्वारा मूल्यांकन एक कार्यात्मक बाह्य मैट्रिक्स के बयान (भूमिकाः द्वारा संकेत के रूप में ) मात्रा।
एक अतिरिक्त लाभ यह है कि उपास्थिकोशिका-हाइड्रोजेल निर्माणों हैनवगठित उपास्थि ऊतक के यांत्रिक गुणों के लिए मूल्यांकन किया जा सकता है। गैरपरिरूद्ध संपीड़न परीक्षण की तुलना के लिए अकोशिकीय हाइड्रोजेल पर किया जाना चाहिए कि ध्यान दें। हाइड्रोजेल, वास्तव में, कारण सीएस moieties की कठोरता के लिए एक आंतरिक कठोरता है। (एक तनाव दर / एस 1% से कम) 5-20% की गैरपरिरूद्ध संपीड़न तनाव लदान शर्त के तहत उपास्थि ऊतक द्वारा अनुभवी शारीरिक तनाव के बाद से उपास्थि ऊतक 11,12 के यांत्रिक परीक्षण के लिए लागू किया जा सकता 10-20 होने की सूचना दी गई है % 13,14। यांत्रिक परीक्षण करने के लिए सेल से लदी और अकोशिकीय दोनों हाइड्रोजेल की प्रतिक्रिया संस्कृति अंत बिंदु पर मूल्यांकन किया गया था। वर्णित उदाहरण में हम OA chondrocytes युक्त निर्माणों के निचले कठोरता के विपरीत वयस्क और किशोर chondrocytes युक्त निर्माणों की एक तुलनीय कठोरता मनाया ऊपर। सेल-हाइड्रोजेल निर्माण के इस तरह के यांत्रिक गुणों के कार्यात्मक संपत्तियों के मूल्यांकन की अनुमतिसेल परिपक्वता की क्षमता का एक में गहराई से विश्लेषण देने का गठन ऊतक।
अंत में, उपास्थि ऊतक उत्पन्न करने के लिए अलग अलग उपास्थिकोशिका आबादी की क्षमता का अध्ययन करने के लिए 3 डी biomimetic हाइड्रोजेल का उपयोग व्यापक रूप से लागू किया जा सकता है। यहाँ वर्णित इन विट्रो अध्ययन में इसके अलावा, सेल से लदी निर्माणों के vivo प्रत्यारोपण शारीरिक संदर्भ में सेल परिपक्वता और पुनर्योजी क्षमता का अध्ययन करने के लिए कल्पना की जा सकती है। अतिरिक्त biomimetic कारकों के साथ हाइड्रोजेल मंच के आगे संशोधन भी उपास्थिकोशिका प्रसार और परिपक्वता का अनुकूलन करने की कल्पना की जा सकती है।
Disclosures
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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