3D هيدروجيل السقالات لالمفصلي خلية غضروفية الثقافة والجيل الغضروف
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
كما ورد في هذا البروتوكول، والهلاميات المائية 3D هي قادرة على الحفاظ على خلية غضروفية النمط الظاهري في الثقافة، وتجنب عملية فقد التمايز الخلوي في خلايا الغضروف الليفي عادة اجه مع الثقافات أحادي الطبقة 15. وعلاوة على ذلك، كشفت الثقافات على المدى الطويل هيدروجيل بناء chondrocytes- بيئة مواتية التي تحافظ على الميزات خلية الجوهرية المرتبطة مع التقدم في السن والمرض.
استخدام هيدروجيل بيوميمتيك 3D ديها العديد من المزايا. أولا، إدراج شوندروتن كبريتات (CS)، عنصرا رئيسيا وجدت في الغضروف المفصلي، تمكن الخلايا من تدهور مصفوفة هيدروجيل عن طريق إفراز chondroitinase ووضع الغضروف تصنيعه حديثا خارج الخلوي مصفوفة 5، 16. وبالإضافة إلى ذلك، فقد تبين CS أن لها خصائص مضادة للالتهاب في المفصل الملتهب. ويمكن أيضا هيدروجيل بيوميمتيك أن تستخدم كمادة سقالات للتسليم خلية في إصلاح الغضروف، ويمكن تعديله كيميائيالتسهيل أفضل الأنسجة بيولوجية التكامل 17،18.
استخدام الهلاميات المائية PEG-CS يسمح الثقافات على المدى الطويل غضروفية وتقييم الخصائص الحيوية والميكانيكية. نحن هنا تظهر كيف يمكن لهذا النظام الأساسي أن يكون مفيدا للتحليلات مقارنة من مصادر مختلفة من الخلايا الغضروفية متباينة من أجل تحديد نوع الخلية الأمثل للهندسة الغضروف. ومن المثير للاهتمام، غضروفية مغلفة في الهلاميات المائية لا تزال قابلة للحياة وتتكاثر وفقا لقدراتهم الذاتية. ويدعم تكوين هيدروجيل، في الواقع، ونمو الأحداث والبالغين غضروفية صحية كما هو مبين في الشكل (2). وتكوينها وهيكلها من الهلاميات المائية وصفها أيضا يعزز تكوين الأنسجة الغضروف كما يتضح من ترسب مصفوفة خارج الخلية الوظيفية المقررة من قبل غلكسمينوغلكن] (GAG ) الكمي.
ميزة إضافية هي أن يبني-خلية غضروفية هيدروجيليمكن تقييمها للالخواص الميكانيكية للأنسجة الغضاريف التي شكلت حديثا. لاحظ أن اختبار ضغط غير محصورة ينبغي أن يقوم على هيدروجيل لا خلوي للمقارنة. الهلاميات المائية، في الواقع، لديهم تصلب الجوهرية بسبب صلابة من الأنصاف CS. سلالة ضغط غير محصورة من 5-20٪ (بمعدل 1٪ سلالة / ثانية) يمكن تطبيقها لاختبار الميكانيكية الغضروف نسيج 11،12 منذ سلالة الفسيولوجية التي يمر بها أنسجة الغضروف تحت ظروف التحميل تم الإبلاغ عن أن تكون 10-20 ٪ 13،14. تم تقييم استجابة كل خلية لادن واخلوي الهلاميات المائية لاختبار الميكانيكية في ثقافة نقطة النهاية. في المثال المذكورة أعلاه لاحظنا صلابة مقارنة للبنيات التي تحتوي على غضروفية الكبار والأحداث على النقيض من صلابة أقل من بنيات التي تحتوي على غضروفية OA. هذه الخواص الميكانيكية للبناء خلايا هيدروجيل تسمح بتقييم الخصائص الفنية للالأنسجة شكلت إعطاء تحليل متعمق من القدرة نضوج الخلية.
في الختام، واستخدام الهلاميات المائية 3D المحاكاة البيولوجية لدراسة إمكانيات السكان خلية غضروفية مختلفة لتوليد أنسجة الغضروف يمكن تطبيقها على نطاق واسع. إلى جانب الدراسات في المختبر الموصوفة هنا، في الجسم الحي زرع يبني خلايا محملة يمكن تصوره لدراسة نضوج الخلايا وإمكانات التجدد في سياق الفسيولوجية. ويمكن أيضا إدخال تعديلات إضافية من منصة هيدروجيل مع العوامل الجزيئية الحيوية إضافية يمكن تصوره لتحسين انتشار خلية غضروفية والنضج.
Divulgations
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 |
References
- Roberts, S., Menage, J., Sandell, L. J., Evans, E. H., Richardson, J. B. Immunohistochemical study of collagen types I and II and procollagen IIA in human cartilage repair tissue following autologous chondrocyte implantation. Knee. 16, 398-404 (2009).
- Smeriglio, P., et al. Comparative Potential of Juvenile and Adult Human Articular Chondrocytes for Cartilage Tissue Formation in Three-Dimensional Biomimetic Hydrogels. Tissue engineering. Part A. , (2014).
- Lai, J. H., Kajiyama, G., Smith, R. L., Maloney, W., Yang, F. Stem cells catalyze cartilage formation by neonatal articular chondrocytes in 3D biomimetic hydrogels. Scientific reports. 3, 3553 (2013).
- Taipale, J., Keski-Oja, J. Growth factors in the extracellular matrix. FASEB journal : official publication of the Federation of American Societies for Experimental Biology. 11, 51-59 (1997).
- Varghese, S., et al. Chondroitin sulfate based niches for chondrogenic differentiation of mesenchymal stem cells. Matrix Biol. 27, 12-21 (2008).
- Park, J. S., et al. The effect of matrix stiffness on the differentiation of mesenchymal stem cells in response to TGF-beta. Biomaterials. 32, 3921-3930 (2011).
- Wang, T., Lai, J. H., Han, L. H., Tong, X., Yang, F. Chondrogenic Differentiation of Adipose-Derived Stromal Cells in Combinatorial Hydrogels Containing Cartilage Matrix Proteins with Decoupled Mechanical Stiffness. Tissue engineering. Part A. , (2014).
- Smith, R. L., et al. Effects of intermittent hydrostatic pressure and BMP-2 on osteoarthritic human chondrocyte metabolism in vitro. J Orthop Res. 29, 361-368 (2011).
- Buschmann, M. D., Gluzband, Y. A., Grodzinsky, A. J., Kimura, J. H., Hunziker, E. B. Chondrocytes in agarose culture synthesize a mechanically functional extracellular matrix. J Orthop Res. 10, 745-758 (1992).
- Farndale, R. W., Buttle, D. J., Barrett, A. J. Improved quantitation and discrimination of sulphated glycosaminoglycans by use of dimethylmethylene blue. Biochim Biophys Acta. 883, 173-177 (1986).
- Lai, J. H., Levenston, M. E. Meniscus and cartilage exhibit distinct intra-tissue strain distributions under unconfined compression. Osteoarthritis and cartilage / OARS, Osteoarthritis Research Society. 18, 1291-1299 (2010).
- Li, L. P., Buschmann, M. D., Shirazi-Adl, A. Strain-rate dependent stiffness of articular cartilage in unconfined compression. Journal of biomechanical engineering. 125, 161-168 (2003).
- Armstrong, C. G., Bahrani, A. S., Gardner, D. L. In vitro measurement of articular cartilage deformations in the intact human hip joint under load. The Journal of bone and joint surgery. American. 61, 744-755 (1979).
- Macirowski, T., Tepic, S., Mann, R. W. Cartilage stresses in the human hip joint. Journal of biomechanical engineering. 116, 10-18 (1994).
- Benya, P. D., Shaffer, J. D. Dedifferentiated chondrocytes reexpress the differentiated collagen phenotype when cultured in agarose gels. Cell. 30, 215-224 (1982).
- Buckwalter, J. A., Mankin, H. J. Articular cartilage: tissue design and chondrocyte-matrix interactions. Instructional course lectures. 47, 477-486 (1998).
- Wang, D. A., et al. Multifunctional chondroitin sulphate for cartilage tissue-biomaterial integration. Nat Mater. 6, 385-392 (2007).
- Simson, J. A., Strehin, I. A., Allen, B. W., Elisseeff, J. H. Bonding and fusion of meniscus fibrocartilage using a novel chondroitin sulfate bone marrow tissue adhesive. Tissue engineering. Part A. 19, 1843-1851 (2013).
