Patent Application
20240124815
A tissue-engineered (TE) construct is a promising treatment for soft tissue injury in the meniscus, ligaments, tendons, neurons, etc. A bioreactor is typically used to manufacture mechanically competent engineered tissue, such as the meniscus construct, by enabling control of both environmental and mechanical conditions. It has been previously shown that tissue-engineered meniscus constructs have significantly higher mechanical properties when cultured in bioreactors under mechanical stimuli such as tensile loading. A bioreactor is required to keep a tissue-engineered construct sterile while scaling up the manufacturing process. However, typical bioreactor systems often require external power sources, making it challenging for the maintenance of the sterile environment. Furthermore, typical bioreactors are heavily customized, making the scale-up of the manufacturing process extremely expensive.
Currently, multiple types of bioreactors are used to promote cell/tissue growth, including spinner flasks, perfusion bioreactors, and mechanical stimulation bioreactors. However, to apply a mechanical force, these bioreactors require that the grafts used to create constructs be taken outside of an incubator. Alternatively, or additionally, these bioreactors require wired connections outside the incubator with a power source and controller. These requirements increase the risk of infection by exposing the grafts to external sources of infection. Alternatively, or additionally, these bioreactors are too small for the creation of tissue-engineered constructs or utilize highly customized electrical and mechanical components.
Accordingly, there is a need to develop a modular and autonomous bioreactor system that can maintain sterility, allow robust mechanical control for the creation of the tissue-engineered constructs, and increase the ability to scale up the manufacturing process of tissue-engineered constructs.
The presently disclosed bioreactor is modular, stackable, and provides mechanical stimulation to a graft to form a tissue-engineered construct. The bioreactor is mechanically stable, chemically stable, and provides a sterile environment for the creating of the tissue-engineered constructs. Thus, the bioreactor disclosed is advantageous over current bioreactors for the creation of tissue-engineered constructs.
The bioreactor includes a movable part and a fixed part, a chamber for positioning a graft for the creation of the tissue-engineered constructs, and a driving system to apply the mechanical stimulation to the graft.
Additional features of the present disclosure will become apparent to those skilled in the art upon consideration of illustrative embodiments exemplifying the best mode of carrying out the disclosure as presently perceived.
The detailed description particularly refers to the accompanying figures in which:
The present disclosure relates to embodiments of a bioreactor system 2 that can be used to create tissue-engineered constructs, including but not limited to ligaments, tendons, and menisci. Specifically, the present disclosure relates to embodiments of a bioreactor system 2 that can be used to provide mechanical stimulation, including but not limited to a tensile force on grafts and/or cells to form tissue-engineered constructs. The bioreactor system includes a bioreactor 10 and a housing 4 comprising a motor and a self-contained power source configured to engage the bioreactor.
As discussed in detail herein, and shown in
The bioreactor system 2 is mechanically stable to withstand forces created during the culture process, especially during clamping and attaching the graft 12 to the bioreactor 10. The ability of the bioreactor system 2 to withstand the changing forces ensures that the graft 12 is not damaged. The bioreactor system 2 is chemically stable even after being placed in a container comprising culture media for a long time, ensuring that chemicals do not adversely affect the graft 12. The bioreactor system 2 allows the entire culture process to be carried out in a sterile environment. The bioreactor system 2 produces a stable and repeatable stretching function. The bioreactor system 2 is configured so that all measurements are accurate and any stretching length error caused by bioreactor design is minimized.
As shown in
Certain exemplary embodiments will now be described to provide an overall understanding of the principles of the disclosure. One or more examples of these embodiments are illustrated in the accompanying drawings.
An exemplary bioreactor 10 illustrated as comprising two parts or components is shown in
In the illustrative example, shown in
As shown in
In the illustrative example, shown in
In the illustrative example, shown in
As shown in
In other embodiments, the bioreactor 310 may include different mechanisms to translate the vertical motion along the axis C to the horizontal movement of the rack along the axis D. For example, the bioreactor may include a jack system, a link system, a bell crank etc.
As shown in
During the stretching function of the worm-gear bioreactor 410, the distance the slide 424 is driven outwards does not uniformly increase with the rotation of the worm structure 440. The process from the beginning of the rotation to the end of the rotation comprises a non-linear stretching process and a linear stretching process. This is because of the slack between the worm structure 440 and the gear 434. Therefore, in some embodiments, the slide 424 may be driven out in advance to avoid the nonlinear process. After the slide 424 is driven out, the grafts 12 may be fixed onto the bioreactor 410. During the linear stretching process, the distance that the slide 424 is driven out for every 45° of rotation on the worm structure 440 may range from about 0.4 mm to about 0.5 mm including any distance or range comprised therein, In some embodiments, the distance that the slide 424 is driven out for every 45° of rotation on the worm structure 440 may be about 0.423 mm.
In other embodiments, different controllers and/or power sources may be used. The electronics 464 (e.g., motor 456, motor driver 458, Arduino Uno microcontroller 460, 9V battery 462) are housed in a housing 462 to protect them from incubator conditions including media, temperature etc. (
In some embodiments, a wireless adapter can be attached to the microcontroller 460, and the motor output signals from the microcontroller 460 can be controlled wirelessly. For example, a wireless adapter like ESP8266 Wifi module can be connected to the microcontroller 460, which can receive and transmit signals wirelessly. Such a configuration can allow multiple bioreactors 410 to be programmed at the same time. In some embodiments, a load cell and a PID controller can be connected to monitor and control the stress that each graft 12 experiences. Alternatively, each microcontroller 460 can be individually programmed to control the corresponding motor 456 in the bioreactor system 450.
In some embodiments, a micro load cell like CZL639HD can be connected to the bioreactor 410 and the microcontroller 460 in order to measure the mechanical stress. The stress data can be used as feedback to adjust the motor control input through a PID controller. Such a feedback loop can keep the stress consistent during the culture period. A user can monitor the data in real time at a remote location when a wireless adaptor is used.
In some embodiments, the bioreactor 410 may further include one or more sensors including but not limited to a pH sensor and/or a micro-Raman/FTIR probe to monitor the pH of the media and the composition of the graft 12 (e.g., amount of collagen in the grafts 12).
In some embodiments, a rechargeable battery with a charging port can be positioned in the housing 452 or a wireless charging technology can be implemented to charge the bioreactor system 450 in the incubator. In some embodiments, the microcontroller 460 can be connected to buttons positioned on the side of the housing 452. The bioreactor system 450 can be programmed to transmit various pre-programmed signals depending on which button was selected by the user. In some embodiments, an automated culture media circulation can be attached to the bioreactor system 450. Such a configuration can allow fresh culture media to be replenished automatically, hence reducing the labor costs.
A method 500 of creating tissue-engineered constructs 14 is shown in
The bone plugs used in this study were prepared according to published methods. Briefly, the bone plugs were extracted from the distal femurs of 1-3-day-old bovids (Gold Medal Packing Inc.) using an electric drill with a 6 mm coring bit. During dissection process, excess tissue around the head and neck of the femur was removed to expose the surface of the trabecular bone, from which bone plugs were extracted. After drilling out 15 mm long cylindrical bone plugs, a stream of high velocity deionized water was used to rinse out residual blood, marrow, and debris from the pore space and keep the white trabecular bone structure. Then bone plugs were washed for 1 hour in PBS with 0.1% (wt/vol) ethylenediaminetetraacetic acid (EDTA) at room temperature. After that, sequential washes in hypotonic buffer (10 mM Trizma base in 100 mL DI water, 0.1 w/v % EDTA) were applied for 1 hour each and at least for three times. Then bone plugs were placed at 4° C. for at least 24 hours. Next, detergent of 10 mM Trizma base with 0.5 w/v % was used for washing bone plugs for at least 3 hours and the bone plugs were room temperature for 24 hours. Finally, the bone plugs were washed using PBS with antibiotics at least seven times until no bubbles were formed.
The collagen used was extracted from rat tails (Pel-Freez Biologicals, Rogers, AZ). The rat tails were immersed in 70% ethanol for 10-20 minutes for thawing. The skin of the rat tail was cut and peeled, and the rat tail tendon was extracted by holding the end of the rat tail and pulling at the tips. After extraction, the collagen was collected into ethanol, the rat tail tendons were dried, and then placed into 0.1% acetic acid. 150 mL of acetic was used per gram of tendon. The collagen was then solubilized for at least 48 hours at 4° C. After that, the collagen solution was placed into 50 mL conical tubes and centrifuged. After being centrifuged at 15000 rpm for 45 minutes at 4° C., the clear supernatant was collected and frozen at below −80° C. for 30 minutes and then lyophilized for 48 hours. Finally, the collagen product was weighed and reconstituted in 0.1% acetic acid solution. The concentration of the collagen was about 30 mg/mL.
Fibrochondrocytes (FCC) were extracted from menisci of 1-3-day-old bovids from the knee joints. Menisci were chopped up into 1-2 mm cubes after being dissected from the joint in sterile environment. After that, cubes were placed into PBS with ABAM (Antibiotic Antimycotic Solution, Mediatch, Inc.), washed three times, and incubated for 30 minutes after the third washing. Then the cubes were added into 0.3% collagenase (Worthington Biochemical Corporation, Lakewood, NJ) solution with 1% ABAM for 18 hours of digestion in a 37° C. incubator with spinning. Then, the digested tissue was pipetted into 100 μm cell strainers into conical tubes to isolate FCCs. After spinning down the conical tubes at 2500 rpm for 12 minutes, the supernatant was aspirated and the cell pellet was dispersed with PBS with 1% ABAM. Conical tubes were used for centrifuging at 1000 rpm for nine minutes. Cell concentrations were adjusted to 150*106 cells/mL in media for graft 12 generation.
The graft 12 comprising a meniscal enthesis construct 12 is composed of bone zone, collagen zone, and an interface between them. The graft 12 is generated from decellularized bone plugs, type I collagen, and fibrochondrocytes prepared as described above. Briefly, marks were made onto a Tygon® tubing to divide zones for collagen and bone. Decellularized bone plugs were placed into the tube at both ends and binder clips were used in avoid of the bone plugs from moving backwards. Holes were cut out at both ends for flowing air during collagen injection. A central hole was cut in the center of the bone zone for injection of collagen. Prepared collagen was mixed with working solution(1 N NaOH, 1× PBS and 10× PBS) and isolated FCCs with media in order to achieve the final collagen mixture with a neutral 7.0 pH, a concentration of 20 mg/mL collagen and 25*106 cells/mL. The mixture was injected into the central hole and the grafts 12 are placed into an incubator under 37° C. for 50 minutes for gelation. After the gelation process, the grafts 12 were removed from the tube into a petri dish with meniscus media and placed in an incubator to equilibrate overnight. Finally, the grafts 12 were clamped onto polysulfone molds with clamps and screws without stretching as a control group, and onto the bioreactor 410 described above as an experimental group to form tissue-engineered constructs 14. Both groups were cultured for the same time periods (2 weeks, 4 weeks). The tissue-engineered constructs 14 were processed for histology analysis and for tensile testing.
For the 2-week culture experiment, the graft 12 were fixed onto polysulfone molds without stretching stimulation as the control group. The experimental group grafts 12 were fixed onto stretching bioreactors 410 with axial stretching stimulation. The stretching rate was 2% of initial collagen length (45° rotation on worm) for every 3 days (5 times of stretching in total). The tissue-engineered constructs 14 from both groups were generated with the same decellularized bone plugs and collagen with FCCs under same conditions. The initial length of collagen was 20 mm (Table 1). After culture, all the tissue-engineered constructs 14 from both groups were processed for Picrosirius red staining and Masson's trichrome staining.
For the 4-week culture experiment, the culture time was changed, and the stretching distance was changed to 8% of initial length of collagen (1.6 mm). Further, the stretching rate was changed from 2% every 3 days to 2% every 6 day. The graft 12 were stretched four times. The other parameters remained unchanged (Table 2). After culture, the collagen length of the tissue-engineered constructs 14 from both groups was measured. Five control group tissue-engineered constructs 14 and four experimental tissue-engineered constructs 14 underwent tensile testing. One control group tissue-engineered constructs 14 and six experimental group tissue-engineered constructs 14 are analyzed with Picrosirius red staining and Masson's trichrome staining. The images of the tissue-engineered constructs 14 placed in containers with media 15 at different time points are shown in
The tissue-engineered constructs 14 were placed into 10% buffered formalin to fix them for 48 hours. After fixing, formalin was washed off with ethanol and the tissue-engineered constructs 14 were longitudinally cut in half to expose their inner structure. Slides of sections were used for Picrosirius red and Masson's trichrome staining. The Picrosirius red staining slides were viewed under brightfield and polarized light with a Nikon Eclipse TE2000-S microscope. The images were taken through a SPOT RT camera.
The 4-week-cultured tissue-engineered constructs 14 were subjected to tensile testing for analysis of mechanical properties. The tensile testing used in this research is based on published methods. Briefly, the tissue-engineered constructs 14 were clamped at both bony ends and vertically fixed onto the testing system (ElectroForce 5500 System, Bose, Eden Prairie, MN). A rate of 1.5 mm/sec was applied to the tissue-engineered constructs 14 until their failure. For mechanical property analysis, several variables were calculated as follow. The ultimate load (UL) was the maximum load that the tissue-engineered constructs 14 can withstand during the testing process. The ultimate tensile stress (UTS) was the maximum stress the tissue-engineered constructs 14 can withstand, and it is the point the tissue-engineered constructs 14 begins to fail. The tensile modulus is the slope of the linear elastic part of stress-strain curve. The toughness is the total area under the stress-strain curve. The strain values were also recorded and analyzed at the onset and at the end of failure.
All the data points in this study are graphed in the form of mean±SD. Data from tensile testing are analyzed with t-testing. Significant difference is determined with p<0.05 and difference existence is determined with 0.05<p<0.1.
The tissue-engineered constructs 14 (meniscal enthesis constructs) were generated from the grafts 12 on polysulfone molds (control groups) and stretching bioreactors (experimental groups). The culture time for the two experiments was respectively 2 weeks and 4 weeks. Following the same generation method, the bone zone 6 and the collagen zone 8 were recognizable from both the control and the experimental groups. The collagen penetrated into the trabecular bone plugs and formed the interface region 7 between the bone zone 6 and the collagen zone 8. The tissue-engineered constructs 14 from both groups were determined to be stiff enough to withstand conventional physical handling without fracture. In the 2-week culture experiment, the observable necking phenomenon was found in samples from the experimental group compared to the control group (
In the 4-week culture experiment, there was no observable necking phenomenon in either group (
Differences in collagen density can be found by comparing brightfield images of Picrosirius red staining slides at the interface region 7 between the unstretched tissue-engineered constructs 14 and the stretched tissue-engineered constructs 14 after 2 weeks of culture (
Similar features also appear in the Picrosirius red staining slides of 4-week tissue-engineered constructs 14. The unstretched tissue-engineered constructs 14 (
After 4 weeks of culture, differences in collagen fiber organization between unstretched and stretched tissue-engineered constructs 14 were observed (
Masson's trichrome staining images demonstrate the cell morphology of FCCs in the tissue-engineered constructs 14. The cells are elongated along the direction of fiber orientations at interface and collagen region, while they remain rounded in shape in the bone region. Both are found in unstretched and stretched tissue-engineered constructs 14, as cells are elongated mainly along the radial direction at the interface region 7 while they change to a longitudinal direction in the collagen region (
The tissue-engineered constructs 14 from the control groups and the experimental groups showed different features during the stretching to failure process. Still shots of the tensile testing process were taken at the same time points for comparison between the control groups and the experimental groups. Necking was first observed in the control tissue-engineered constructs 14 (
After collecting and analyzing the tensile testing data, the stress-strain curves of the tissue-engineered constructs 14 from both groups were examined. Obvious differences were observed by comparing curves from control groups and experimental groups. After calculation, the values of different mechanical properties are listed in Table 3. Comparison of UL, UTS, tensile modulus, and strain of failure are compared (
By comparing the stress-strain curves from control groups and experimental groups, it was observed that most of the unstretched tissue-engineered constructs 14 showed higher ultimate load (UL), ultimate tensile stress (UTS), and tensile modulus compared to the stretched tissue-engineered constructs 14. However, the stress-strain curves of stretched tissue-engineered constructs 14 generally showed the feature of higher strain after the onset of failure during tensile testing compared with control tissue-engineered constructs 14 (
The figures provided herein are not necessarily to scale, although a person skilled in the art will recognize instances where the figures are to scale and/or what a typical size is when the drawings are not to scale. Additionally, a number of terms may be used throughout the disclosure interchangeably but will be understood by a person skilled in the art. Further, to the extent features, sides, or steps are described as being “first” or “second,” such numerical ordering is generally arbitrary, and thus such numbering can be interchangeable. Lastly, the present disclosure includes some illustrations and descriptions that include prototypes or bench models. A person skilled in the art will recognize how to rely upon the present disclosure to integrate the techniques, systems, devices, and methods provided for into a product in view of the present disclosures.
This application claims priority to U.S. Provisional Application Ser. No. 63/415,605, filed 12 Oct. 2022, which is expressly incorporated by reference herein.
| Number | Date | Country | |
|---|---|---|---|
| 63415605 | Oct 2022 | US |
