An experimental technique for the treatment of osteochondral defects in the rabbit’s knee joint is described. The implantation of allogeneic mesenchymal stem cells into osteochondral defects provides a promising development in the field of tissue engineering. The preparation of fibrin-cell-clots in vitro offers a standardized method for implantation.
The treatment of osteochondral articular defects has been challenging physicians for many years. The better understanding of interactions of articular cartilage and subchondral bone in recent years led to increased attention to restoration of the entire osteochondral unit. In comparison to chondral lesions the regeneration of osteochondral defects is much more complex and a far greater surgical and therapeutic challenge. The damaged tissue does not only include the superficial cartilage layer but also the subchondral bone. For deep, osteochondral damage, as it occurs for example with osteochondrosis dissecans, the full thickness of the defect needs to be replaced to restore the joint surface 1. Eligible therapeutic procedures have to consider these two different tissues with their different intrinsic healing potential 2. In the last decades, several surgical treatment options have emerged and have already been clinically established 3-6.
Autologous or allogeneic osteochondral transplants consist of articular cartilage and subchondral bone and allow the replacement of the entire osteochondral unit. The defects are filled with cylindrical osteochondral grafts that aim to provide a congruent hyaline cartilage covered surface 3,7,8. Disadvantages are the limited amount of available grafts, donor site morbidity (for autologous transplants) and the incongruence of the surface; thereby the application of this method is especially limited for large defects.
New approaches in the field of tissue engineering opened up promising possibilities for regenerative osteochondral therapy. The implantation of autologous chondrocytes marked the first cell based biological approach for the treatment of full-thickness cartilage lesions and is now worldwide established with good clinical results even 10 to 20 years after implantation 9,10. However, to date, this technique is not suitable for the treatment of all types of lesions such as deep defects involving the subchondral bone 11.
The sandwich-technique combines bone grafting with current approaches in Tissue Engineering 5,6. This combination seems to be able to overcome the limitations seen in osteochondral grafts alone. After autologous bone grafting to the subchondral defect area, a membrane seeded with autologous chondrocytes is sutured above and facilitates to match the topology of the graft with the injured site. Of course, the previous bone reconstruction needs additional surgical time and often even an additional surgery. Moreover, to date, long-term data is missing 12.
Tissue Engineering without additional bone grafting aims to restore the complex structure and properties of native articular cartilage by chondrogenic and osteogenic potential of the transplanted cells. However, again, it is usually only the cartilage tissue that is more or less regenerated. Additional osteochondral damage needs a specific further treatment. In order to achieve a regeneration of the multilayered structure of osteochondral defects, three-dimensional tissue engineered products seeded with autologous/allogeneic cells might provide a good regeneration capacity 11.
Beside autologous chondrocytes, mesenchymal stem cells (MSC) seem to be an attractive alternative for the development of a full-thickness cartilage tissue. In numerous preclinical in vitro and in vivo studies, mesenchymal stem cells have displayed excellent tissue regeneration potential 13,14. The important advantage of mesenchymal stem cells especially for the treatment of osteochondral defects is that they have the capacity to differentiate in osteocytes as well as chondrocytes. Therefore, they potentially allow a multilayered regeneration of the defect.
In recent years, several scaffolds with osteochondral regenerative potential have therefore been developed and evaluated with promising preliminary results 1,15-18. Furthermore, fibrin glue as a cell carrier became one of the preferred techniques in experimental cartilage repair and has already successfully been used in several animal studies 19-21 and even first human trials 22.
The following protocol will demonstrate an experimental technique for isolating mesenchymal stem cells from a rabbit’s bone marrow, for subsequent proliferation in cell culture and for preparing a standardized in vitro-model for fibrin-cell-clots. Finally, a technique for the implantation of pre-established fibrin-cell-clots into artificial osteochondral defects of the rabbit’s knee joint will be described.
A. Preparation of a Donor Rabbit for the Isolation of Mesenchymal Stem Cells (Surgery Room)
B. Flushing of Rabbit MSC from Bones and Expansion (Cell Culture Hood)
C. Preparation of Fibrin Clots in vitro
D. Implantation of Allogeneic Mesenchymal Stem Cells in Fibrin Clots
The described surgical technique permits a successful isolation and implantation of allogeneic mesenchymal stem cells into an artificial osteochondral defect. The experimental setup resulted in a successful integration of the implant into the surrounding cartilage.
The defect was filled by repair tissue with similar biomechanical properties and similar durability compared to the surrounding cartilage. The fibrin-cell-clot was prepared in vitro on a sterile plate with pre-drilled holes, which had the same size as the osteochondral defect (Figure 3). As a result, there were no clefts between the implanted fibrin clot and the surrounding cartilage, which would be a risk factor for premature degeneration or delamination (Figure 6). A basal healing of the repair tissue was ensured because the subchondral bone was penetrated, and thus worked against a shearing. Another important aspect was the stiffness of the repair tissue, which should match the healthy surrounding cartilage tissue to avoid an increased load on it and a possible premature degeneration. In our preliminary experiments (data not shown), we showed that after 12 weeks a sufficient rigidity could already be achieved. Moreover, an intact and homogeneous surface of the transplant was found, which reduced shear stress and possible implant damage (Figure 6).
Figure 1. Separation of blood cells and plasma from PBMCs and MSCs using Biocoll Separating Solution.
Figure 2. Monolayer of mesenchymal stem cells (New Zealand White rabbit) after 5 days in culture.
Figure 3. Sterile plate with pre-drilled holes (3×3.6 mm) the top one being filled with a fibrin-cell-clot.
Figure 4. Opened knee joint after medial parapatellar arthrotomy.
Figure 5. Osteochondral defects (3 mm deep, 3 mm in diameter, figure eight-shaped) in the trochlear groove.
Figure 6. Opened knee joint 12 weeks after implantation of two fibrin-cell-clots into two drilled osteochondral defects.
In recent years, the possibility of treating complex articular osteochondral defects – such as those resulting from osteochondritis dissecans, osteonecrosis and trauma – with Tissue Engineering approaches became more and more attractive. In the previously mentioned pathologic entities, tissue damage extends to the subchondral bone and involves two tissues characterized by different intrinsic healing capacities 1. There is an increasing interest in the role of subchondral bone for the pathogenic processes of osteochondral articular damage 11,23. The functional conditions of articular cartilage and its supporting bone are tightly connected. Injuries of either tissue adversely affect the mechanical environment as well as the homeostatic balance of the entire joint 24. Alterations in the osteochondral unit through mechanical disruption of joint motion, loose body formation, mechanical wear in the involved compartment and attrition of opposing surfaces may lead to an earlier onset and development of osteoarthritis 1,11. Therefore, tissue engineering approaches for the regeneration of osteochondral defects should be accompanied by an adequate restoration of the underlying subchondral bone in order to enhance the effective union with surrounding host tissues 2. Mesenchymal stem cells seem to be an ideal cell source to provide these specific requirements of osteochondral repair. The protocol highlights the promotion of bone and cartilage tissue restoration by potentially inducing the selective differentiation of the transplanted mesenchymal stem cells in osteogenic and chondrogenic lineages.
In comparison to chondrocytes, mesenchymal stem cells have several major advantages: they can be easily isolated from bone marrow, synovialis and fat tissue without any greater donor side morbidity. Mesenchymal stem cells do not differentiate during in vitro expansion and therefore can be culture-expanded in large numbers to treat large articular cartilage defects. Moreover, they seem to be immunosuppressive and – in response to appropriate stimuli – can differentiate into chondrocytes and osteocytes 25,26. Another remarkable advantage of mesenchymal stem cells displays their hypoimmunity, which means that allogeneic mesenchymal stem cells can be used without any sign of rejection reaction 27. Therefore, a cell-pool from one or two donor animals can be sufficient for all experiments. This reduces operation time and additional harm to the animals.
Several experiments showed promising results using fibrin glue for osteochondral repair 19,20. Usually, the inoculation of the fibrinogen-cell-suspension with thrombin solution has been described as a procedure done in situ, directly into the artificial osteochondral lesions. After a short period of time of pre-clotting (after a few minutes) the operation is usually finished by relocating the patella and wound closure.
In several pilot tests, we found out that for sufficient clotting of the fibrin-cell-suspension it takes more than 60 min. In situ – during the operation – it is hardly possible to wait more than 60 min for entire clotting. Moreover, by use of a sterile plate with pre-drilled holes simulating the osteochondral lesions, it was possible to show that the amount of fibrin glue, which was used to obviously fill the defect completely, was not enough due to shrinking of the hardening clot. This requires a higher volume of fibrin glue in advance in order to achieve a congruent and complete filling of the defect. Performing the preparation of the clot in vitro it is possible to easily adjust the construct shape to the appropriate size of the drilled defect and therefore, fill the osteochondral defect totally and congruently.
Additionally, an in vitro preparation of the cell clots prevents a leakage of the adhesive but (after only a few minutes) not fully hardened fibrin-cell-suspension. Therefore, it can be guaranteed that the initially intended volume will stay in the defect and will begin with cartilage integration and remodeling.
The described technique permits a standardized method for experimental stem cell research in the field of osteochondral repair. The protocol provides a reproducible way to isolate mesenchymal stem cells in order to re-implant them later in osteochondral cartilage defects of the rabbit’s knee joint. Autologous chondrocytes have already been implanted into osteochondral defects in a fibrin-cell-model 19. Using an in vitro-model of clot preparation as well as mesenchymal stem cells instead of chondrocytes appears to be a more advantageous and promising new approach for remodeling and repair of osteochondral lesions.
The authors have nothing to disclose.
This project was funded by the German Research Association (grant HE 4578/3-1) and partially by the FP7 EU-Project “GAMBA” NMP3-SL-2010-245993.
Name of reagent/equipment | Company | Catalogue Number | Comments |
DMEM | Biochrom AG | F 0415 | |
FCS | PAN Biotech GmbH | 0401 | |
Propofol | Fresenius Kabi | ||
Penicillin/Streptomycin | Biochrom AG | A 2210 | 1,000 units/10 μg/μl in 0.9% NaCl |
PBS Dulbecco (1X) | Biochrom AG | L1815 | |
Ethanol (70%) | Merck KGaA | 410230 | |
Trypan Blue Solution (0.4%) | Sigma-Aldrich | T8154 | |
Biocoll Separation Sol. | Biochrom AG | L6115 | Isotonic solution Density: 1,077 g/ml |
Trypsin-EDTA 0.05% | Invitrogen GmbH | 25300-054 | |
Fentanyl | DeltaSelectGmBH | 1819340 | |
NaCl solution (0.9%) | BBraun | 8333A193 | |
Syringes (Injekt) | BBraun | 4606108V | |
Needles (Sterican) | BBraun | 4657519 | |
Forceps (blunt/sharp) | Aesculap | ||
Scissors | Aesculap | ||
Scalpels | Feather Safety Razor Co | 02.001.30.022 | |
Pipettes research | Eppendorf | ||
Bone Cutter | Aesculap | ||
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 |
CO2 Incubator | Forma Scientific Inc. | ||
Cell culture laminar flow hood Hera Safe | Heraeus Instruments | ||
Sterile saw | Aesculap | ||
Centrifuge Megafuge 2.0 R | Heraeus Instruments | ||
Hemocytometer | Brand GmbH+Co KG | 717810 | Neubauer |
Air operated power drill | Aesculap | ||
TISSUCOL-Kit 1.0 ml Immuno | Baxter | 2546648 | |
Fibers (4-0 Monocryl, 4-0 Vicryl) | Ethicon | ||
Spray dressing (OpSite) | Smith&Nephew | 66004978 | Permeable for water vapor |