Applying a Three-dimensional Uniaxial Mechanical Stimulation Bioreactor System to Induce Tenogenic Differentiation of Tendon-Derived Stem Cells

Summary

A three-dimensional uniaxial mechanical stimulation bioreactor system is an ideal bioreactor for tenogenic-specific differentiation of tendon-derived stem cells and neo-tendon formation.

Abstract

Tendinopathy is a common chronic tendon disease relating to inflammation and degeneration in an orthopaedic area. With high morbidity, limited self-repairing capacity and, most importantly, no definitive treatments, tendinopathy still influences patients’ life quality negatively. Tendon-derived stem cells (TDSCs), as primary precursor cells of tendon cells, play an essential role in both the development of tendinopathy, and functional and structural restoration after tendinopathy. Thus, a method that can in vitro mimic the in vivo differentiation of TDSCs into tendon cells would be useful. Here, the present protocol describes a method based on a three-dimensional (3D) uniaxial stretching system to stimulate the TDSCs to differentiate into tendon-like tissues. There are seven stages of the present protocol: isolation of mice TDSCs, culture and expansion of mice TDSCs, preparation of stimulation culture medium for cell sheet formation, cell sheet formation by culturing in stimulation medium, preparation of 3D tendon stem cell construct, assembly of the uniaxial-stretching mechanical stimulation complex, and evaluation of the mechanical stimulated in vitro tendon-like tissue. The effectiveness was demonstrated by histology. The entire procedure takes less than 3 weeks. To promote extracellular matrix deposition, 4.4 mg/mL ascorbic acid was used in the stimulation culture medium. A separated chamber with a linear motor provides accurate mechanical loading and is portable and easily adjusted, which is applied for the bioreactor. The loading regime in the present protocol was 6% strain, 0.25 Hz, 8 h, followed by 16 h rest for 6 days. This protocol could mimic cell differentiation in the tendon, which is helpful for the investigation of the pathological process of tendinopathy. Moreover, the tendon-like tissue is potentially used to promote tendon healing in tendon injury as an engineered autologous graft. To sum up, the present protocol is simple, economic, reproducible and valid.

Introduction

Tendinopathy is one of the common sports injuries. It is mainly manifested by pain, local swelling, decreased muscle tension in the affected area, and dysfunction. The incidence of tendinopathy is high. The presence of Achilles tendinopathy is most common for middle- and long-distance runners (up to 29%), while the presence of patellar tendinopathy is also high in athletes of volleyball (45%), basketball (32%), track and field (23%), handball (15%), and soccer (13%)^1,2,3,4,5. However, due to the limited self-healing ability of the tendon, and the lack of effective treatments, tendinopathy still influences patients’ life negatively^6,7. Moreover, the pathogenesis of tendinopathy remains unclear. There have been many investigations about its pathogenesis, mainly including "inflammation theory", "degeneration theory", "overuse theory", and so forth⁸. At present, many researchers believed that tendinopathy was due to the failed self-repair to the micro-injuries caused by excessive mechanical loading the tendon experiences^9,10.

Tendon-derived stem cells (TDSCs), as primary precursor cells of tendon cells, play an essential role in both development of tendinopathy and functional and structural restoration after tendinopathy^11,12,13. It was reported that mechanical stress stimulation could cause the proliferation and differentiation of osteocytes, osteoblasts, smooth muscle cells, fibroblasts, mesenchymal stem cells and other force-sensitive cells^{14,15,16,17,18}. Therefore, TDSCs, as one of the mechanosensitive and multipotent cells, can similarly be stimulated to differentiate by mechanical loading^19,20.

However, different mechanical loading parameters (loading strength, loading frequency, loading type and loading period) can induce TDSCs to differentiate into different cells²¹. Thus, an effective and valid mechanical loading regime is very significant for tenogenesis. Furthermore, there are different kinds of bioreactors as stimulation systems currently used for providing mechanical loading to TDSCs. The principles of each kind of bioreactor are different, so the mechanical loading parameters corresponding to different bioreactors are also different. Therefore, a simple, economic, and reproducible stimulation protocol is in demand, including the type of bioreactor, the corresponding stimulation medium, and the mechanical loading regime.

The present article describes a method based on a three-dimensional (3D) uniaxial stretching system to stimulate the TDSCs to differentiate into tendon-like tissue. There are seven stages of the protocol: isolation of mice TDSCs, culture and expansion of mice TDSCs, preparation of stimulation culture medium for cell sheet formation, cell sheet formation by culturing in stimulation medium, preparation of 3D tendon stem cell construct, assembly of the uniaxial-stretching mechanical stimulation complex, and evaluation of the mechanical stimulated in vitro tendon-like tissue. The whole procedure takes less than 3 weeks to obtain the 3D cell construct, which is far less than some existing methods^22,23. The present protocol has been proven to be able to induce TDSCs to differentiate into tendon tissue, and it is more reliable than the current commonly used two-dimensional (2D) stretching system²¹. The effectiveness was demonstrated by histology. In short, the present protocol is simple, economic, reproducible and valid.

Protocol

The methods described were approved and performed in accordance with the guidelines and regulations of the University of Western Australia Animal Ethics Committee.

1. Isolation of mice TDSCs

1. Euthanize the 6-8-week-old C57BL/6 mice by cervical dislocation.
2. Harvest patellar tendons²⁴ and Achilles tendons²⁵.
3. Digest tendons from one with 6 mL of type I collagenase (3 mg/mL) for 3 h.
   NOTE: As the size of tendon in the mouse is small, all tendons harvested from one mouse should be used in this step.
4. Collect the cells and culture in complete Minimal Essential Medium (Alpha-MEM) containing 10% of fetal bovine serum (FBS) and 1% of streptomycin and penicillin mixture for 7 days.
5. Identify TDSCs using fluorescence-activated cell sorting by flow cytometry (expression of cell surface markers including CD44, CD90 and Sca-1; lack of the expression of CD34 and CD45).
6. Passage and freeze cells (passage 4 cells will be used for further steps).

2. Culture and expansion of mice TDSCs

NOTE: Conduct all steps in a sterile biosafety hood.

1. Take the warmed-up cells (mice TDSCs, 1 million cells, passage 4, to 37 °C).
2. Slowly add extra 4-5 mL of complete Minimal Essential Medium (Alpha-MEM).
3. Transfer the mixture to a 15 mL centrifuge tube with a pipette.
4. Top up with medium to 8 mL total with a pipette.
   1. Prewarm medium in a 37 °C oven incubator.
5. Place the tube into centrifuge and balance.
6. Centrifuge and pellet cells at 347 x g for 5 min.
7. Take out after centrifuge and check the pellet at the bottom.
8. Decant the freezing medium, and resuspend cells gently in 1-2 mL of complete medium (avoid making too many bubbles).
9. Transfer resuspended cells to a T-75 flask by a pipette.
10. Use a pipette to add complete Alpha-MEM Medium to the flask to reach a total volume of 10 mL with final concentration of 13,000 cells/cm².
11. Place the flask into the incubator and culture at 37 °C with 5% CO₂.
12. Change the medium every 3 days.
13. Observe and monitor the cells under a microscope when changing the medium until cells are cultured to 100% confluence (about 40,000 cells/cm²).

3. Preparation of stimulation culture medium for cell sheet formation

NOTE: Conduct all steps in a sterile biosafety hood.

1. Pour 15 mL of complete Alpha-MEM medium into a 15 mL sterile tube.
2. Add 6 µL of ascorbic acid (11 mg/mL) into 15 mL of medium for a final concentration of 4.4 µg/mL.
   NOTE: Additionally adding 25 ng/mL connective tissue growth factor in the stimulation culture medium can accelerate the whole growth and differentiation process.
3. Mix gently by inverting up and down.

4. Cell sheet formation by culturing in stimulation medium

NOTE: Conduct all steps in a sterile biosafety hood.

1. Discard the normal complete alpha-MEM medium carefully, and avoid touching the cells attached to the bottom of the flask.
2. Add 10 mL of stimulation medium slowly and avoid causing any disturbance to the fully confluent cells.
3. Culture the cells in stimulation medium for 9 days (change the medium every 3 days) to sufficiently generate the cell sheet at 37 °C with 5% CO₂.
   NOTE: Extracellular matrix will become thick and present to be cloudy when observed from the bottom of the flask after stimulated by ascorbic acid, which means the cell sheet is sufficiently generated.

5. Preparation of 3D tendon stem cell construct

NOTE: Conduct all steps in a sterile biosafety hood.

1. Take the flask out of the incubator.
2. Discard the stimulation medium completely.
3. Wash the monolayer cell sheet with phosphate buffer saline (PBS) by swirling the flask.
4. Discard the PBS completely.
5. Use a pipette to add 1 mL of 0.25% trypsin to the corner of the flask.
6. Tap the corner of the flask to de-attach the monolayer cell sheet until the corner of the cell sheet is off the bottom of the flask.
7. Immediately add 9 mL of complete Alpha-MEM medium to stop the trypsinization.
8. Continue swirling the flask to peel off the monolayer cell sheet completely.
9. Pour the total de-attached cell sheet into a Petri dish with medium.
10. Use a sterile tweezer to pick up one corner of the cell sheet and rotate in a clockwise direction for 15 times.
11. Pick up another end of the cell sheet and rotate in an anti-clockwise direction for 10 times to firmly generate a tendon-like in vitro construct (Figure 1A).

6. Assembly of the uniaxial-stretching mechanical stimulation complex in unique designed bioreactor

NOTE: Conduct all steps in a sterile biosafety hood.

1. Connect the hooks by the connecter and adjust to the desired distance (2 cm) between two hooks.
2. Gently wind the 3D TDSCs construct on the assembled hook for 3 times on each hook (Figure 1B).
3. Secure the hooks with cell construct onto the chamber of the bioreactor by tightening the screws on two ends.
   NOTE: The whole chamber, including screws and hooks, is sterile (autoclave for 1.5 h at 134 °C and ultraviolet rays (UV) expose for 24 hours before use). For the metal holder of chambers, UV light expose for 48 hours before use.
4. Fill up the chamber with complete Alpha-MEM medium.
5. Connect the actuator to the culture chamber by cable.
6. Cut the hooks connecter by sterile scissors.
7. Switch on the power and corresponding channel controller to start mechanical stimulation.
8. Put the lid on the chamber.
9. Check the indicator lights and assure the bioreactor can function properly.
10. Put the bioreactor in the incubator and subject the 3D cell construct to mechanical stretching for 6 days (6% stretching, 0.25 Hz, 8 h, followed by 16 h rest).

7. Evaluation of the mechanical stimulated in vitro tendon-like tissue

1. Loosen the screws by a screwdriver and take the assembly off carefully.
2. Put the tendon-like tissue into 4% paraformaldehyde for fixation for 15 min.
3. Place the fixed tendon-like tissue into a biopsy cassette with biopsy foam pads.
4. Process to dehydrate the sample and finally evaluate by H&E histological staining.
5. Repeat the whole protocol and extract RNA from the tendon-like tissue for evaluating the expression of tenogenic markers by quantitative PCR (qPCR). Primers for the selected genes are listed in Table 1.
   NOTE: Histology protocol and qPCR protocol are standard and following a previous study²¹.

Results

Before mechanical stimulation, TDSCs were grown to 100% confluence in complete medium and displayed a disorganized ultrastructural morphology (Figure 2A). After 6 days of uniaxial stretching mechanical loading, extracellular matrix (ECM) and cell alignments were well orientated (Figure 2B). Cells were well populated and well enveloped in ECM after mechanical loading. Cell morphology was presented to be elongated and was more similar to normal tendon cell compared to the one without stret...

Discussion

The tendon is a mechanosensitive fibrous connective tissue. According to previous research, excess mechanical loading could lead to osteogenic differentiation of tendon stem cells, whereas insufficient loading would lead to disordered collagen fiber structure during tendon differentiation²¹.

A common view is that the key to an ideal bioreactor is the ability to simulate the in vitro cellular microenvironment that cells in vivo undergo. Therefore, mimicking the in vivo n...

Materials

Name	Company	Catalog Number	Comments
Ascorbic acid	Sigma-aldrich	PHR1008-2G
Fetal bovine serum (FBS)	Gibcoä by Life Technologies	1908361
Histology processor	Leica	TP 1020
Minimal Essential Medium (Alpha-MEM)	Gibcoä by Life Technologies	2003802
Mouse Tendon Derived Stem Cell			Isolated from Achilles tendons of 6- to 8-wk-old C57BL/6 mice. Then digested with type I collagenase (3 mg/ml; MilliporeSigma, Burlington, MA, USA) for 3 h and passed through a 70 mmcell strainer to yield single-cell suspensions.
Paraformaldehyde	Sigma-aldrich	441244
Streptomycin and penicillin mixture	Gibcoä by Life Technologies	15140122
Three-dimensional Uniaxial Mechanical Stimulation Bioreactor System	Centre of Orthopaedic Translational Research, Medical School, University of Western Australia		Available from the corresponding author upon request. Or make it according to our design* *Wang T, Lin Z, Day RE, et al. Programmable mechanical stimulation influences tendon homeostasis in a bioreactor system. Biotechnol Bioeng. 2013;110(5):1495–1507. doi:10.1002/bit.24809
Trypsin	Gibcoä by Life Technologies	1858331