We designed a novel mechanical loading bioreactor that can apply uniaxial or biaxial mechanical strain to a cartilage biocomposite prior to transplantation into an articular cartilage defect.
We designed a loading device that is capable of applying uniaxial or biaxial mechanical strain to a tissue engineered biocomposites fabricated for transplantation. While the device primarily functions as a bioreactor that mimics the native mechanical strains, it is also outfitted with a load cell for providing force feedback or mechanical testing of the constructs. The device subjects engineered cartilage constructs to biaxial mechanical loading with great precision of loading dose (amplitude and frequency) and is compact enough to fit inside a standard tissue culture incubator. It loads samples directly in a tissue culture plate, and multiple plate sizes are compatible with the system. The device has been designed using components manufactured for precision-guided laser applications. Bi-axial loading is accomplished by two orthogonal stages. The stages have a 50 mm travel range and are driven independently by stepper motor actuators, controlled by a closed-loop stepper motor driver that features micro-stepping capabilities, enabling step sizes of less than 50 nm. A polysulfone loading platen is coupled to the bi-axial moving platform. Movements of the stages are controlled by Thor-labs Advanced Positioning Technology (APT) software. The stepper motor driver is used with the software to adjust load parameters of frequency and amplitude of both shear and compression independently and simultaneously. Positional feedback is provided by linear optical encoders that have a bidirectional repeatability of 0.1 μm and a resolution of 20 nm, translating to a positional accuracy of less than 3 μm over the full 50 mm of travel. These encoders provide the necessary position feedback to the drive electronics to ensure true nanopositioning capabilities. In order to provide the force feedback to detect contact and evaluate loading responses, a precision miniature load cell is positioned between the loading platen and the moving platform. The load cell has high accuracies of 0.15% to 0.25% full scale.
We have designed a loading bioreactor that is capable of applying uniaxial or biaxial mechanical strain to a tissue engineered biocomposites fabricated for transplantation. This device is primarily designed as a bioreactor for engineered replacements for articular cartilage; it could also be used for other load-bearing tissues in the human body. Our motivation in this bioreactor design stems from Drachman and Sokoloff 1, who made the seminal observation of abnormal formation of articular cartilage in paralyzed chick embryos due to absence of motion. Similarly, physical exercise is essential for development of normal muscle and bone. In keeping with this concept, many research groups have investigated how different modes of physical stimuli during in vitro cultivation modulates the biochemical and mechanical properties of cell-biomaterial biocomposites and tissue explants 2-7. The concept of functional tissue engineering involves the in vitro use of mechanical stimuli to enhance the functional properties of tissues, i.e. the mechanical properties that enable the tissue to withstand the expected in vivo stress and strain 8,9. Numerous studies report the use mechanical loading in terms of shear and compression to stimulate engineered cartilage constructs for articular joints. Mauck et al. 10 suggest that mechanical loading alone can induce chondrogenesis of mesenchymal stem cells even in the absence of growth factors that are considered vital. Application of intermittent mechanical loading such as compression or shear during tissue cultivation has been shown to modulate cartilage and bone formation, however the optimum dosimetry of loading differs with cell and tissue properties 11.
The most important function of articular cartilage is the ability to withstand compressive and shear forces within the joint, therefore it has to have high compressive and shear moduli. The lack of functional mechanical strength and physiological ultrastructure in engineered cartilage has resulted in the breakdown on neo-cartilage in vivo and the failure of cartilage replacement strategies in joints. Although compression and shear have been commonly demonstrated to modulate and improve mechanical strength of articular cartilage biocomposites, a combination approach is rare 6,12-15. Wartella and Wayne 16 designed a bioreactor that applied tension and compression to produce meniscal cartilage replacements. Waldman et al. 15 designed a device to apply compression and shear to chondrocytes cultured in a porous calcium polyphosphate substrate. Bian et al. 17 demonstrated mechanical properties matching native cartilage with the in vitro cultivation of adult canine chondrocytes in gels and application of biaxial mechanical loading (compressive deformational loading and sliding contact loading).
The biaxial mechanical loading bioreactor was originally designed by Danielle Chu in our laboratory with the overall goal to induce morphological adaptations in tissue engineered cartilage constructs resulting in higher compressive and shear moduli than currently available 18. We believe this research will significantly increase our broader understanding of how mechanotransduction can be modulated to engineer clinically relevant tissues.
1. Biaxial Loading Bioreactor Design
2. Cell-Seeded Agarose Constructs
3. Culture the Disks
4. Immobilization of Samples for Mechanical Loading
5. Mechanical Loading
6. Calibrating Loading Platen
To ensure that the proper strains are applied to samples, each platen must be carefully calibrated prior to starting an experiment.
7. Writing a Dosing Protocol
In this study we define compressive and shear strain as follows:
Example Biaxial Dosing Protocol
Sample thickness: 2.25 mm
Tare Strain (Compression): 10% of the sample thickness (0.225 mm)
Dynamic Strain Amplitude (Compression): 10% (+/- 5% of the sample thickness)
Frequency (Compression): 1 Hz
Dynamic Strain Amplitude (Shear): 25% of the sample thickness (0.5625 mm): Shear stress is
applied to the sample by the platen moving horizontally.
Frequency (Shear): 0.5 Hz
Typical dosing protocol is 3 hr of loading per day.
In this example, dynamic and shear loading is applied simultaneously rather than sequentially. We believe this pattern better mimics the complex loading environment in the human knee.
Difference from Calibration Value | Vertical Position | |
Platen Calibration Value (touches bottom of bioreactor) | 0 mm | 29.7700 mm |
Platen makes contact with Sample (2.25 mm sample) | 4.4140 mm | 25.3560 mm |
Strain (5% Thickness ) | 4.3015 mm | 25.4705 mm |
Strain (10% Thickness ) | 4.1890 mm | 25.5810 mm |
Strain (15% Thickness ) | 4.0765 mm | 25.6955 mm |
The device was tested by using agarose gels seeded with 20 million cells/ml chondrocytes and cultivated in the presence of uniaxial (compression) or biaxial (compression and shear) mechanical loading. Primary porcine chondrocytes were isolated from the articular cartilage of 2-4 month old pigs. 5 mm diameter and 1.5 mm thick samples were cultured in 2 ml of defined chondrogenic culture medium (High glucose DMEM, 1% ITS+ Premix, 100 U/ml penicillin, 100 μg/ml streptomycin, 2 mM L-glutamine, 2.5 μg/ml amphotericin B, 50 μg/ml ascorbic acid, 0.1 mM nonessential amino acids (NEAA), 0.4 mM proline,) in 24-well plates at 37 °C, 5%CO2. 10-7 M dexamethasone and 10 ng/ml TGF-β1 were supplied for the first 10 days of culture. The samples were loaded for 3 hr/day between days 10-30. Uniaxial loading consisted of 10% compression peak-to-peak amplitude, 1 Hz and biaxial loading consisted of 0.15 mm (10% thickness) compression and 0.075 mm shear peak-to-peak amplitude, 1 Hz. The dynamic strain amplitude and loading frequency are chosen based on published studies 17,19. At the end of 30 days biochemical and mechanical properties of engineered cartilage were assessed.
This study employed three groups: 1- No loading control, 2- Uniaxial (compressive) loading, 3- Biaxial (compressive and shear) loading. The DNA contents and the wet weights of constructs remained similar in the three groups after 30 days of cultivation (p > 0.05). The GAG content was highest in the group that was subjected to biaxial loading (group 3, p < 0.001 compared with the control group), followed by the uniaxial loading group (group 2, p < 0.05) (Figure 6 ). The GAG contents of groups 2 and 3 correspond to 48% and 50% of native cartilage, respectively. Group 3 resulted in significantly higher amount of collagen than groups 1 and 2 (p < 0.01). Group 2 also had thicker constructs than group 1 (p < 0.01). Surprisingly, the equilibrium compressive Young’s modulus was the highest in group 2 (uniaxial loading, p < 0.01) and there were no significant differences between group 3 and 1. The Young’s modulus of group 2 corresponded to 60.1% of native porcine cartilage.
The histological analyses indicated positive and homogeneous staining for glycosaminoglycans (alcian blue, safranin O) and type II collagen (Figure 7). All groups stained negative for type I collagen (not shown).
In summary, these preliminary results suggest that this bioreactor successfully applied compression and biaxial (compression and shear) mechanical loading during long term cultivation of engineered tissues. In this study biaxial loading was shown to the proteoglycan and collagen deposition and the thickness of the tissue engineered cartilage samples. Uniaxial compression increased both the proteoglycan deposition and the Young’s modulus.
Figure 1. Biaxial loading is accomplished by the X-stage (shearing) and the Z-stage(compression). The figure shows a custom-made loading platen attached to the stages to load samples in a 24-well plate. The loading parameters are controlled with a computer connected to the stepper motors 18.
Figure 2. Left: The polysulfone loading platen designed for 24-well plates. Right: The loading platen attachment to the biaxial loading bioreactor.
Figure 3. Preparation of agarose wells for immobilizing samples during shear loading. The immobilized construct placed in the agarose well for mechanical loading. This figure shows a 1.5 mm thick agarose well and a 2.25 mm thick sample.
Figure 4. The graphical user interface to control the biaxial loading device. Click here to view larger figure.
Figure 5. Example Biaxial Loading Move Sequence Program Graphical User Interface: Dynamic Compression Move Sequence Program (10% tare compression, 10% dynamic strain amplitude, 1 Hz) and Dynamic Shear Move Sequence Program (10% tare compression, 25% dynamic shear strain amplitude, 0.5 Hz). Click here to view larger figure.
Figure 6. Biochemical and mechanical testing results (n=6). ***p < 0.001, **p < 0.01, *p < 0.05 compared with group 1 (unloaded control. Group 2: Uniaxial compressive loading, group 3: Biaxial compressive and shear loading.
Figure 7. Histology: Alcian blue/nuclear fast red staining, Safranin O/fast green, immunochemistry for type II collagen.
We have designed a loading device that is capable of applying uniaxial or biaxial mechanical strain to tissue engineered constructs fabricated for transplantation. The device can be used as a bioreactor for in vitro cultivation of engineered biocomposites or as a testing device to describe the mechanical characteristics of the native tissue or after other treatments prior to. The device subjects engineered tissue constructs to biaxial mechanical loading with great precision of loading dose (amplitude and frequency) and application to a wide variety of tissue culture conditions from single to 24 well plates.
The application of shear loading presented a set of unique challenges for the design of this system. To maximize nutrient transfer, constructs were originally unconfined in individual wells of a 24 well plate. This did not present a problem for dynamic compression, as the tare compressive strain insured that sample-platen contact was not lost. When shear strain was added to the protocol, however, unconfined samples slid along the bottom of the plate and some lost contact with the platen. Additionally, during biaxial loading protocols samples had the tendency to flip over, causing inconsistent loading. We solved this problem by creating the agarose wells to immobilize samples as described in the procedure. These agarose wells allow consistent biaxial loading of samples without limiting nutrient availability to samples.
Unlike the compression bioreactors that are very widely researched 20,21, our device is capable of applying precise strains on multiple axes. These axes can be independently controlled. Multiaxial loading can be applied sequentially or simultaneously. It is possible to implement a third Y-axis to provide mechanical loading in three dimensions to better mimic in vivo conditions.
While other multiaxial bioreactors have been developed to mimic the mechanical environment of the joint, they have large limitations compared to our system. A shear and compression apparatus designed by Frank, et al. allows up to 12 samples to be loaded simultaneously with load feedback, however constructs are not confined or secured 6. During experiments involving shear strain, it is essential that constructs be secured so that they do not slide under the loading platen. Sliding will result in uneven and inconsistent shear loading of the specimen. Newer bioreactors, such as the unique “rolling ball” system 22,23 and a biaxial stimulation device 16, create a much more realistic and consistent loading environment; however, they only allow one sample to be loaded at a time. Large sample sizes are essential for performing the necessary biochemical, mechanical, and histological analyses on the constructs with a high level of confidence. Additionally, the “rolling ball” system lacks force feedback, an essential measure of construct development during long term in vitro cultivation. It also allows the prevention of platen-specimen non-contact and specimen overloading, which will irreversibly damage tissue engineered constructs. A sliding contact bioreactor developed by Bian, et al. allows up to four constructs to be loaded simultaneously, but still lacks this valuable force feedback mechanism 17.
The current setup using 24-well plates allows the simultaneous loading of 24 samples; more samples are possible with modifications of the geometry of the loading platen. The loading platen offers vast flexibility to the novel design. The chosen material polysulfone is porous, can be sterilized and cultivated in the humid and warm environment of an incubator. It is easily machinable, enabling a variety of geometries and numbers of samples to be loaded simultaneously.
In conclusion, the new biaxial loading bioreactor for tissue engineering enables long term in vitro cultivation of tissue engineered constructs. Biaxial loading increased the proteoglycan and collagen deposition and the thickness of the tissue engineered cartilage samples but did not seem to significantly influence the mechanical properties of engineered cartilage as we hypothesized. Uniaxial compression increased both the proteoglycan deposition and the Young’s modulus. We believe that the optimum dose of mechanical loading differs with cell and tissue properties. Future studies of collagen architecture and dosimetry of loading will allow us to fully evaluate the effects of biaxial loading on the development of engineered tissues.
The authors have nothing to disclose.
This work was supported by the Office of Research and Development, RR&D Service, U.S. Department of Veterans Affairs, NIH COBRE 1P20RR024484, NIH K24 AR02128 and Department of Defense W81XWH-10-1-0643.
REAGENTS | |||
DMEM, High glucose, pyruvate | Invitrogen | 11995 | |
Agarose Type II | Sigma | CAS 39346-81-1 | |
Penicillin Streptomycin Glutamine 100X | Invitrogen | 10378-016 | |
ITS+ Premix | BD Biosciences | 354352 | |
Pen Strep Glutamine | Invitrogen | 10378-016 | |
Amphotericin B | Invitrogen | 041-95780 | |
Ascorbic Acid | Sigma | A-2218 | |
Nonessential Amino Acid Solution 100x | Sigma | M-7145 | |
L-proline | Sigma | P-5607 | |
Dexamethasone | Sigma | D-2915 | |
Recombinant Human Transforming Growth Factor β1 | R&D Systems | 240-B-010 | |
EQUIPMENT | |||
Model 31 Load Cell (1000 g) | Honeywell | AL311 | |
Single Channel Display | Honeywell | SC500 | |
50 mm Linear Encoded Travelmax Stage with Stepper Actuator | Thorlabs | LNR50SE/M | |
Two Channel Stepper Motor Controller | Thorlabs | BSC102 | |
50 mm Trapezoidal Stepper Motor Drive (2) | Thorlabs | DRV014 | |
Adjustable Kinematic Locator (4) | Thorlabs | KL02 | |
Precision Right Angle Plate | Thorlabs | AP90/M | |
Vertical Mounting Bracket | Thorlabs | LNR50P2/M | |
Solid Aluminum Breadboard | Thorlabs | MB3030/M | |
Gel Casting System with 1.5 mm and 0.75 mm spacer plates | BioRad | #1653312 and #1653310 | |
Disposable Biopsy Punch, 5 mm | Miltex, Inc. | 33-35 | |
16 mm hollow punch | Neiko Tools | ||
Non-Tissue Culture Treated Plates, 24 Well, Flat Bottom | BD Biosciences | 351147 | |
Ultra-Moisture-Resistant Polysulfone sheet for loading platens | McMaster-Carr | 86735k19 | Custom-machined |