3D системы для культивирования правам суставные хондроциты в синовиальной жидкости
Summary
3D системы культивирования человеческих хондроцитов суставного к высоким уровням синовиальной жидкости описывается. Синовиальная жидкость отражает наиболее естественный микросреду для суставного хряща, и может быть легко получена и сохранена. Эта система при этом может быть использована для изучения регенерации хряща и для скрининга терапии для лечения артрита.
Abstract
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%) 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%) 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.
Protocol
Discussion
В этом докладе, мы разработали метод, который позволяет культуре человеческих хондроцитов суставного в 3D среде в среде, содержащей высокие концентрации человеческого синовиальной жидкости. Синовиальная жидкость является одним из основных компонентов, составляющих природной среды в …
Disclosures
The authors have nothing to disclose.
Acknowledgements
Мы хотели бы поблагодарить Робина Най (Тафтс Медицинский центр), Tomoya Uchimura и Дана Кэрнс (Университет Тафта) за оказание помощи в синовиальной жидкости хранения и центрифугирования. Эта работа финансировалась NIH (1R01AR059106-01A1) для LZ
Materials
Table of specific reagents and equipment:
| Name of the Reagent | Company | Catalogue Number | Comments |
|---|---|---|---|
| Alginate (Alginic Acid sodium salt) | Sigma | A2158-250G | 2.4% solution stored at 40°C |
| Calcium Chloride Dihydrate, Granular | J.T. Baker | A19339 | |
| Chondrogenic Growth media | Lonza | CC-3156 (base media) | |
| CC-4409 (supplement) | |||
| Chondrogenic Differentiation Media | Lonza | CC-3226 (base media) | |
| CC-4408 (supplement) | |||
| Human articular chondrocytes | Lonza | CC-2550 | |
| Dapi (4′,6-Diamidino-2-phenylindole dihydrochloride) | Sigma-Aldrich | D9542 | |
| RNeasy mini kit (for RNA extraction) | Qiagen | 74104 | |
| PCR reagents: SYBR-green | Quanta | 95053-500 | |
| 12 ml syringe | Tyco-Kendall-Monoject | 512852 | |
| 22-Gague Hypodermic Needle | Tyco-Kendall-Monoject | 8881 | |
| Microscope | Olympus | IX71 | |
| Platform rocker | Thermoscientific thermolyne | Vari-mix | |
| Primers sequences | |||
| Collagen IIa-forward | 5′-TTC ATC CCA CCC TCT CAC AGT-3′ | ||
| Collagen IIa-reverse | 5′-CCTCTGCCTTGACCCGAA-3′ | ||
| MMP13-forward | 5′-TGT GCC CTT CTT CAC ACA GAC ACT-3′ | ||
| MMP13-reverse | 5′-GAG AGC AGA CTT TGA GTC ATT GCC-3′ | ||
| Caspase 3-forward | 5′-TCA TTA TTC AGG CCT GCC GTG GTA-3′ | ||
| Caspase 3-reverse | 5′-TGG ATG AAC CAG GAG CCA TCC TTT -3′ |
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