Name: applying a three-dimensional uniaxial mechanical stimulation bioreactor system to induce tenogenic differentiation of tendon-derived stem cells
Uploaded: 2020-08-01T14:00:00
Description: Watch this Scientific Journal Video about Applying a Three-dimensional Uniaxial Mechanical Stimulation Bioreactor System to Induce Tenogenic Differentiation of Tendon-Derived Stem Cells at JoVE.com

The research was carried out while the author was in receipt of &#8220;a University of Western Australia International Fee Scholarship and a University Postgraduate Award at The University of Western Australia&#8221;. This work was supported by National Natural Science Foundation of China (81802214).

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
<ol>
	<li>Euthanize the 6-8-week-old C57BL/6 mice by cervical dislocation.</li>
	<li>Harvest patellar tendons24 and Achilles tendons25.</li>
	<li>Digest tendons from one with 6 mL of type I collagenase (3 mg/mL) for 3 h. 
	NOTE: As the size of tendon i...

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...

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&#8217; life negatively6,7. Moreover, the pathogenesis of tendinopathy remains unclear. There have been many investigations about its pathogenesis, mainly including &#34;inflammation theory&#34;, &#34;degeneration theory&#34;, &#34;overuse theory&#34;, 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 experiences9,10.
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 tendinopathy11,12,13. 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 cells14,15,16,17,18. Therefore, TDSCs, as one of the mechanosensitive and multipotent cells, can similarly be stimulated to differentiate by mechanical loading19,20.
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 methods22,23. 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.

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	<tbody>
		<tr height="156">
			<td height="156" style="height:156px;width:240px;">Ascorbic acid</td>
			<td style="width:213px;">Sigma-aldrich</td>
			<td style="width:119px;">PHR1008-2G</td>
			<td style="width:288px;"></td>
		</tr>
		<tr height="130">
			<td height="130" style="height:130px;">Fetal bovine serum (FBS)</td>
			<td>Gibco&#228; by Life Technologies</td>
			<td align="right">1908361</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:240px;">Histology processor</td>
			<td style="width:213px;">Leica</td>
			<td style="width:119px;">TP 1020</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Minimal Essential Medium (Alpha-MEM)</td>
			<td>Gibco&#228; by Life Technologies</td>
			<td align="right" style="width:119px;">2003802</td>
			<td></td>
		</tr>
		<tr height="126">
			<td height="126" style="height:126px;width:240px;">Mouse Tendon Derived Stem Cell</td>
			<td style="width:213px;"></td>
			<td style="width:119px;"></td>
			<td style="width:288px;">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.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Paraformaldehyde</td>
			<td style="width:213px;">Sigma-aldrich</td>
			<td style="width:119px;">441244</td>
			<td style="width:288px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Streptomycin and penicillin mixture</td>
			<td>Gibco&#228; by Life Technologies</td>
			<td align="right">15140122</td>
			<td></td>
		</tr>
		<tr height="168">
			<td height="168" style="height:168px;width:240px;">Three-dimensional Uniaxial Mechanical Stimulation Bioreactor System</td>
			<td style="width:213px;">Centre of Orthopaedic Translational Research, Medical School, University of Western Australia</td>
			<td style="width:119px;"></td>
			<td style="width:288px;">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&#8211;1507. doi:10.1002/bit.24809</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:240px;">Trypsin</td>
			<td>Gibco&#228; by Life Technologies</td>
			<td style="width:119px;">1858331</td>
			<td></td>
		</tr>
	</tbody>
</table>

applying a three-dimensional uniaxial mechanical stimulation bioreactor system to induce tenogenic differentiation of tendon-derived stem cells

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&#8217; 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.

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...

Watch this Scientific Journal Video about Applying a Three-dimensional Uniaxial Mechanical Stimulation Bioreactor System to Induce Tenogenic Differentiation of Tendon-Derived Stem Cells at JoVE.com

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

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.

Tendinopathy is a common chronic tendon disease relating to inflammation and degeneration in an orthopaedic area. With high morbidity, limited ...

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