An experimental technique for the treatment of chondral defects in the rabbit’s knee joint is described. The implantation of autologous chondrocytes seeded on a matrix is a well-accepted method for the remodeling and repair of articular cartilage lesions providing satisfying long-term results. Matrix-assisted autologous chondrocyte transplantation (MACT) offers a standardized and clinically established implantation method.
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.
A. Cartilage Biopsy (Surgery Room; Steps 1-5 in Non-sterile Preparation Room)
B. In vitro Culture
C. Cell-seeding of the Matrix
D. Matrix-assisted Autologous Chondrocyte Transplantation
The described surgical technique permits a successful isolation and implantation of autologous chondrocytes into an artificial chondral defect. The experimental setup resulted in a successful integration of the implant into the surrounding cartilage.
After 12 weeks in vivo, the chondral defect was filled by repair tissue with a homogenous and intact surface, which reduced shear stress and damage to the implant (Figure 4). Moreover, no hypertrophy or calcification of the implant was seen. The repair tissue showed a stiff and solid quality, which was comparable to the healthy surrounding cartilage tissue. This was an important aspect because increased load on the adjacent cartilage due to insufficient biomechanical properties of the implant would be a risk for premature degeneration. Moreover, a graft delamination has not occurred. Having cut the membrane to the same size of the defect preoperatively, any fissuring or clefts between implant and surrounding cartilage were avoided.
Figure 1. Monolayer of chondrocytes primary culture at 80% confluency.
Figure 2. Taking a cartilage biopsy out of the femoral trochlea groove.
Figure 3. Artificially created trochlear cartilage defect.
Figure 4. Opened knee joint 12 weeks after implantation of a membrane seeded with autologous chondrocytes into a pre-drilled chondral defect (red arrow).
The presented protocol provides an established 9,12,13 and easily reproducible technique to isolate autologous chondrocytes for subsequent proliferation and re-implantation into artificially created cartilage defects in rabbit knees. The use of autologous chondrocytes for remodeling and repair of articular cartilage lesions is already in clinical use providing satisfying long-term results 6.
Major problems as for example periosteal hypertrophy and calcification, graft delamination or donor site morbidity occurred with the first and second generation of autologous chondrocyte implantation 14. Therefore researchers have developed techniques using exogenous bioresorbable materials to deliver chondrocytes to the defect site. The positive effect of three-dimensional culture system on the maintenance of the chondrocytic characteristics has been repeatedly demonstrated, for example when using agarose 15, poly – dioxanon and polyglactin 16, polyesters poly(L-lactide) (PLLA) and poly(D,L-lactide-coglycolide) (PLGA) 17, fibrin 12, hyaluronan 18, alginate 19, collagen 20 or collagen-matrices 9.
The bilayer collagen membrane Chondro-Gide used in this study has been successfully applied in several preclinical studies before 9,21,22 and is already part of clinical applications 21,23. The bilayer structure of the collagen membrane offers a stable microenvironment for chondrocyte integration and proliferation, allows for an even distribution of the cells within the matrix and eliminates the possibility of cell leakage as it is seen in conventional ACI 11. By use of this MACT technique, the transplanted chondrocytes are locally retained with maintained viability. Matrix-assisted autologous chondrocyte transplantation leads to the synthesis of cartilage-like regeneration tissue and is clinically applicable 21.
The described animal model is well accepted in the experimental treatment of articular cartilage lesions 11,24,25. However, the translation to clinical routine of possible results of experiments using the presented technique is difficult for several reasons. In an animal model it is almost impossible to achieve non or partial weight-bearing in the first days/weeks after surgery which would be generally recommended to allow the implanted cells to start with early integration processes. Full weight bearing immediately after surgery could potentially harm the transplant and therefore might influence the outcome. But this setting was similar in all animal models comparable 9,12. Experiments with larger animals, more closely resembling the human situation, are warranted in further animal studies.
In summary, however, an established experimental animal model as it is described above provides a basis for further experimental studies of cartilage repair and might even facilitate the realization and performance of complex study settings. Experimental examinations with growth factors, gene induction and changing matrix properties using this animal model might be helpful to improve the established clinical settings.
The authors have nothing to disclose.
This project was funded by the German Research Association (DFG, HE 4578/3-1).
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 |