Матрица-Assisted Аутологичный трансплантации хондроцитов для реконструкции и ремонта хрящевые дефекты в модели кролика
Summary
Экспериментальной методики для лечения дефектов хряща в коленном суставе кролика описано. Имплантация аутологичных хондроцитов высевали на матрице это общепринятый метод для реконструкции и ремонта повреждений суставного хряща обеспечивать удовлетворяя долгосрочные результаты. Матрица-Assisted аутологичных хондроцитов трансплантации (МАСТ) предлагает стандартизированные и клинически подтвержденной метод имплантации.
Abstract
Articular cartilage defects are considered a major health problem because articular cartilage has a limited capacity for self-regeneration 1. Untreated cartilage lesions lead to ongoing pain, negatively affect the quality of life and predispose for osteoarthritis. During the last decades, several surgical techniques have been developed to treat such lesions. However, until now it was not possible to achieve a full repair in terms of covering the defect with hyaline articular cartilage or of providing satisfactory long-term recovery 2-4. Therefore, articular cartilage injuries remain a prime target for regenerative techniques such as Tissue Engineering. In contrast to other surgical techniques, which often lead to the formation of fibrous or fibrocartilaginous tissue, Tissue Engineering aims at fully restoring the complex structure and properties of the original articular cartilage by using the chondrogenic potential of transplanted cells. Recent developments opened up promising possibilities for regenerative cartilage therapies.
The first cell based approach for the treatment of full-thickness cartilage or osteochondral lesions was performed in 1994 by Lars Peterson and Mats Brittberg who pioneered clinical autologous chondrocyte implantation (ACI) 5. Today, the technique is clinically well-established for the treatment of large hyaline cartilage defects of the knee, maintaining good clinical results even 10 to 20 years after implantation 6. In recent years, the implantation of autologous chondrocytes underwent a rapid progression. The use of an artificial three-dimensional collagen-matrix on which cells are subsequently replanted became more and more popular 7-9.
MACT comprises of two surgical procedures: First, in order to collect chondrocytes, a cartilage biopsy needs to be performed from a non weight-bearing cartilage area of the knee joint. Then, chondrocytes are being extracted, purified and expanded to a sufficient cell number in vitro. Chondrocytes are then seeded onto a three-dimensional matrix and can subsequently be re-implanted. When preparing a tissue-engineered implant, proliferation rate and differentiation capacity are crucial for a successful tissue regeneration 10. The use of a three-dimensional matrix as a cell carrier is thought to support these cellular characteristics 11.
The following protocol will summarize and demonstrate a technique for the isolation of chondrocytes from cartilage biopsies, their proliferation in vitro and their seeding onto a 3D-matrix (Chondro-Gide, Geistlich Biomaterials, Wollhusen, Switzerland). Finally, the implantation of the cell-matrix-constructs into artificially created chondral defects of a rabbit’s knee joint will be described. This technique can be used as an experimental setting for further experiments of cartilage repair.
Protocol
Representative Results
Discussion
Представленный протокол обеспечивает установленный 9,12,13 и легко воспроизводимые техники, чтобы изолировать аутологичных хондроцитов для последующего распространения и повторной имплантации в искусственно созданные дефекты хряща кролика в коленях. Использование аутологичны?…
Divulgazioni
The authors have nothing to disclose.
Acknowledgements
Этот проект был профинансирован немецкой ассоциации исследований (DFG Его 4578/3-1).
Materials
| Name of reagent/equipment | Company | Catalogue Number | Comments |
| DMEM | Biochrom AG | F 0415 | |
| Collagenase A | Roche | 10 103 586 001 | 0.21 U/mg |
| Fetal calf serum (FCS) | PAN Biotech GmbH | 3702-P103009 | |
| Propofol | Fresenius Kabi | ||
| Penicillin/Streptomycin | Biochrom AG | A 2213 | 10,000 U/ml/10,000 μg/ml |
| PBS Dulbecco (1X) | Biochrom AG | L1815 | |
| Ethanol (70%) | Merck KgaA | 410230 | |
| Trypsin-EDTA 0.25 %/0.02 % | Biochrom AG | L2163 | in PBS w/o Ca2+, Mg2+ |
| Fentanyl | Delta Select GmBH | 1819340 | |
| NaCl solution (0.9%) | Bbraun | 8333A193 | |
| Tissue culture dishes 100 mm/150 mm | TPP AG | 93100/93150 | Growth area 60.1 mm2/147.8 mm2 |
| Tissue culture flasks 25/75 mm2 | TPP AG | 90025/90075 | 25 mm2, 75 mm2 |
| Centrifuge Tubes (50 ml) | TPP AG | 91050 | Gamma-sterilized |
| Hemocytometer | Brand GmbH+Co KG | 717810 | Neubauer |
| Trypan Blue Solution 0.4% | Sigma-Aldrich | L8154 | |
| Spray dressing (OpSite) | Smith&Nephew | 66004978 | Permeable for water vapor |
| Chondro-GideÒ | Geistlich Pharma AG | 30915.5 | |
| Biopsy Punch | pfm medical ag | 48351 | |
| Tissucol Duo S | Baxter | 3419627 | 0.5 ml |
Riferimenti
- Albrecht, C., et al. Gene expression and cell differentiation in matrix-associated chondrocyte transplantation grafts: a comparative study. Osteoarthritis Cartilage. 19, 1219-1227 (2011).
- Pridie, K. H. A method of resurfacing osteoarthritic knee joints. J. Bone Joint Surg. Br. 41, 618-619 (1959).
- Johnson, L. L. Arthroscopic abrasion arthroplasty historical and pathologic perspective: present status. Arthroscopy. 2, 54-69 (1986).
- Steadman, J. R., Rodkey, W. G., Singelton, S. B., Briggs, K. K. Microfracture technique for full-thickness chondral defects: technique and clinical result. Operat. Tech. Orthop. 7, 300-304 (1997).
- Brittberg, M., et al. Treatment of deep cartilage defects in the knee with autologous chondrocyte transplantation. N. Engl. J. Med. 331, 889-895 (1994).
- Peterson, L., Vasiliadis, H. S., Brittberg, M., Lindahl, A. Autologous chondrocyte implantation: a long-term follow-up. Am. J. Sports Med. 38, 1117-1124 (2010).
- Nehrer, S., et al. Chondrocyte-seeded collagen matrices implanted in a chondral defect in a canine model. Biomaterials. 19, 2313-2328 (1998).
- Frenkel, S. R., Toolan, B., Menche, D., Pitman, M. I., Pachence, J. M. Chondrocyte transplantation using a collagen bilayer matrix for cartilage repair. J. Bone. Joint Surg. Br. 79, 831-836 (1997).
- Salzmann, G. M., et al. The dependence of autologous chondrocyte transplantation on varying cellular passage, yield and culture duration. Biomaterials. 32, 5810-5818 (2011).
- Frohlich, M., Malicev, E., Gorensek, M., Knezevic, M., Kregar Velikonja, N. Evaluation of rabbit auricular chondrocyte isolation and growth parameters in cell culture. Cell Biol. Int. 31, 620-625 (2007).
- Willers, C., Chen, J., Wood, D., Xu, J., Zheng, M. H. Autologous chondrocyte implantation with collagen bioscaffold for the treatment of osteochondral defects in rabbits. Tissue Eng. 11, 1065-1076 (2005).
- Vogt, S., et al. The influence of the stable expression of BMP2 in fibrin clots on the remodelling and repair of osteochondral defects. Biomaterials. 30, 2385-2392 (2009).
- Ueblacker, P., et al. In vivo analysis of retroviral gene transfer to chondrocytes within collagen scaffolds for the treatment of osteochondral defects. Biomaterials. 28, 4480-4487 (2007).
- Marlovits, S., Zeller, P., Singer, P., Resinger, C., Vecsei, V. Cartilage repair: generations of autologous chondrocyte transplantation. Eur. J. Radiol. 57, 24-31 (2006).
- Benya, P. D., Shaffer, J. D. Dedifferentiated chondrocytes reexpress the differentiated collagen phenotype when cultured in agarose gels. Cell. 30, 215-224 (1982).
- Rudert, M., Hirschmann, F., Wirth, C. J. Growth behavior of chondrocytes on various biomaterials. Orthopade. 28, 68-75 (1999).
- Hsu, S. H., et al. Evaluation of biodegradable polyesters modified by type II collagen and Arg-Gly-Asp as Tissue Engineering scaffolding materials for cartilage regeneration. Artificial Organs. 30, 42-55 (2006).
- Brun, P., Cortivo, R., Zavan, B., Vecchiato, N., Abatangelo, G. In vitro reconstructed tissues on hyaluronan-based temporary scaffolding. J. Mater. Sci. Mater. Med. 10, 683-688 (1999).
- Domm, C., Fay, J., Schunke, M., Kurz, B. Redifferentiation of dedifferentiated joint cartilage cells in alginate culture. Effect of intermittent hydrostatic pressure and low oxygen partial pressure. Orthopade. 29, 91-99 (2000).
- Kimura, T., Yasui, N., Ohsawa, S., Ono, K. Chondrocytes embedded in collagen gels maintain cartilage phenotype during long-term cultures. Clin. Orthop. Relat. Res. , 231-239 (1984).
- Kon, E., et al. Second-generation autologous chondrocyte implantation: results in patients older than 40 years. Am. J. Sports Med. 39, 1668-1675 (2011).
- Gavenis, K., Schmidt-Rohlfing, B., Mueller-Rath, R., Andereya, S., Schneider, U. In vitro comparison of six different matrix systems for the cultivation of human chondrocytes. In Vitro Cell Dev. Biol. Anim. 42, 159-167 (2006).
- Niemeyer, P., et al. Characteristic complications after autologous chondrocyte implantation for cartilage defects of the knee joint. Am. J. Sports Med. 36, 2091-2099 (2008).
- Tay, L. X., et al. Treatment outcomes of alginate-embedded allogenic mesenchymal stem cells versus autologous chondrocytes for the repair of focal articular cartilage defects in a rabbit model. The American Journal of Sports Medicine. 40, 83-90 (2012).
- Brittberg, M., Nilsson, A., Lindahl, A., Ohlsson, C., Peterson, L. Rabbit articular cartilage defects treated with autologous cultured chondrocytes. Clin. Orthop. Relat. Res. , 270-283 (1996).
