Name: applying a three-dimensional uniaxial mechanical stimulation bioreactor system to induce tenogenic differentiation of tendon-derived stem cells
Uploaded: 2020-08-01T14:00:00.000+00:00
Duration: 14 min 4 s
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 in the mouse is small, all tendons harvested from one mouse should be used in this step.</li>
	<li>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.</li>
	<li>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).</li>
	<li>Passage and freeze cells (passage 4 cells will be used for further steps).</li>
</ol>
2. Culture and expansion of mice TDSCs
NOTE: Conduct all steps in a sterile biosafety hood.
<ol>
	<li>Take the warmed-up cells (mice TDSCs, 1 million cells, passage 4, to 37 &#176;C).</li>
	<li>Slowly add extra 4-5 mL of complete Minimal Essential Medium (Alpha-MEM).</li>
	<li>Transfer the mixture to a 15 mL centrifuge tube with a pipette.</li>
	<li>Top up with medium to 8 mL total with a pipette.
	<ol>
		<li>Prewarm medium in a 37 &#176;C oven incubator.</li>
	</ol>
	</li>
	<li>Place the tube into centrifuge and balance.</li>
	<li>Centrifuge and pellet cells at 347 x g for 5 min.</li>
	<li>Take out after centrifuge and check the pellet at the bottom.</li>
	<li>Decant the freezing medium, and resuspend cells gently in 1-2 mL of complete medium (avoid making too many bubbles).</li>
	<li>Transfer resuspended cells to a T-75 flask by a pipette.</li>
	<li>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.</li>
	<li>Place the flask into the incubator and culture at 37 &#176;C with 5% CO2.</li>
	<li>Change the medium every 3 days.</li>
	<li>Observe and monitor the cells under a microscope when changing the medium until cells are cultured to 100% confluence (about 40,000 cells/cm2).</li>
</ol>
3. Preparation of stimulation culture medium for cell sheet formation
NOTE: Conduct all steps in a sterile biosafety hood.
<ol>
	<li>Pour 15 mL of complete Alpha-MEM medium into a 15 mL sterile tube.</li>
	<li>Add 6 &#181;L of ascorbic acid (11 mg/mL) into 15 mL of medium for a final concentration of 4.4 &#181;g/mL. 
	NOTE: Additionally adding 25 ng/mL connective tissue growth factor in the stimulation culture medium can accelerate the whole growth and differentiation process.</li>
	<li>Mix gently by inverting up and down.</li>
</ol>
4. Cell sheet formation by culturing in stimulation medium
NOTE: Conduct all steps in a sterile biosafety hood.
<ol>
	<li>Discard the normal complete alpha-MEM medium carefully, and avoid touching the cells attached to the bottom of the flask.</li>
	<li>Add 10 mL of stimulation medium slowly and avoid causing any disturbance to the fully confluent cells.</li>
	<li>Culture the cells in stimulation medium for 9 days (change the medium every 3 days) to sufficiently generate the cell sheet at 37 &#176;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.</li>
</ol>
5. Preparation of 3D tendon stem cell construct
NOTE: Conduct all steps in a sterile biosafety hood.
<ol>
	<li>Take the flask out of the incubator.</li>
	<li>Discard the stimulation medium completely.</li>
	<li>Wash the monolayer cell sheet with phosphate buffer saline (PBS) by swirling the flask.</li>
	<li>Discard the PBS completely.</li>
	<li>Use a pipette to add 1 mL of 0.25% trypsin to the corner of the flask.</li>
	<li>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.</li>
	<li>Immediately add 9 mL of complete Alpha-MEM medium to stop the trypsinization.</li>
	<li>Continue swirling the flask to peel off the monolayer cell sheet completely.</li>
	<li>Pour the total de-attached cell sheet into a Petri dish with medium.</li>
	<li>Use a sterile tweezer to pick up one corner of the cell sheet and rotate in a clockwise direction for 15 times.</li>
	<li>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).</li>
</ol>
6. Assembly of the uniaxial-stretching mechanical stimulation complex in unique designed bioreactor
NOTE: Conduct all steps in a sterile biosafety hood.
<ol>
	<li>Connect the hooks by the connecter and adjust to the desired distance (2 cm) between two hooks.</li>
	<li>Gently wind the 3D TDSCs construct on the assembled hook for 3 times on each hook (Figure 1B).</li>
	<li>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 &#176;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.</li>
	<li>Fill up the chamber with complete Alpha-MEM medium.</li>
	<li>Connect the actuator to the culture chamber by cable.</li>
	<li>Cut the hooks connecter by sterile scissors.</li>
	<li>Switch on the power and corresponding channel controller to start mechanical stimulation.</li>
	<li>Put the lid on the chamber.</li>
	<li>Check the indicator lights and assure the bioreactor can function properly.</li>
	<li>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).</li>
</ol>
7. Evaluation of the mechanical stimulated in vitro tendon-like tissue
<ol>
	<li>Loosen the screws by a screwdriver and take the assembly off carefully.</li>
	<li>Put the tendon-like tissue into 4% paraformaldehyde for fixation for 15 min.</li>
	<li>Place the fixed tendon-like tissue into a biopsy cassette with biopsy foam pads.</li>
	<li>Process to dehydrate the sample and finally evaluate by H&#38;E histological staining.</li>
	<li>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.</li>
</ol>

The authors have nothing to disclose.

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.

<table><tbody><tr><td>Ascorbic acid</td><td>Sigma-aldrich</td><td>PHR1008-2G</td><td></td></tr><tr><td>Fetal bovine serum (FBS)</td><td>Gibco&amp;auml; by Life Technologies</td><td>1908361</td><td></td></tr><tr><td>Histology processor</td><td>Leica</td><td>TP 1020</td><td></td></tr><tr><td>Minimal Essential Medium (Alpha-MEM)</td><td>Gibco&amp;auml; by Life Technologies</td><td>2003802</td><td></td></tr><tr><td>Mouse Tendon Derived Stem Cell</td><td></td><td></td><td>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><td>Paraformaldehyde</td><td>Sigma-aldrich</td><td>441244</td><td></td></tr><tr><td>Streptomycin and penicillin mixture</td><td>Gibco&amp;auml; by Life Technologies</td><td>15140122</td><td></td></tr><tr><td>Three-dimensional Uniaxial Mechanical Stimulation Bioreactor System</td><td>Centre of Orthopaedic Translational Research, Medical School, University of Western Australia</td><td></td><td>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&amp;ndash;1507. doi:10.1002/bit.24809</td></tr><tr><td>Trypsin</td><td>Gibco&amp;auml; by Life Technologies</td><td>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 ...

applying-three-dimensional-uniaxial-mechanical-stimulation-bioreactor

Research

JoVE Journal

Medicine

5.8K Views.  University of Western Australia. Verschillende mechanische belastingparameters kunnen pees-afgeleide STAMcellen veroorzaken die in verschillende cellen worden onderscheiden. We introduceren een gedetailleerd proces van de toepassing van deze driedimensionale uniaxiale mechanische stimulatie bioreactor systeem om tenogene differentiatie van pees-afgeleide STAM cellen induceren. Tendinopathie is een van de meest voorkomende sportblessures in de orthopedische afdeling.De aanwezigheid van achilles tendinopathie komt het m...

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

Mechanical Stimulation of Stem Cells Using Cyclic Uniaxial Strain

The role of mechanical forces in the development and maintenance of biological tissues is well documented, including several mechanically regulated phenomena such as bone remodeling, muscular hypertrophy, and smooth muscle cell plasticity. However, the forces involved are often extremely complex and difficult to monitor and control in vivo. To better investigate the effects of mechanical forces on cells, we have developed an in vitro method for applying uniaxial cyclic tensile strain to adherent cells cultured on elastic membranes. This method utilizes a custom-designed bioreactor with a motorized cam-rotor system to apply the desired force. Here we present a step-by-step video protocol demonstrating how to assemble the various components of each "stretch chamber", including, in this case, a silicone membrane with micropatterned topography to orient the cells with the direction of the strain. We also describe procedures for sterilizing the chambers, seeding cells onto the membrane, latching the chamber into the bioreactor, and adjusting the mechanical parameters (i.e. magnitude and rate of strain). The procedures outlined in this particular protocol are specific for seeding human mesenchymal stem cells onto silicone membranes with 10 &micro;m wide channels oriented parallel to the direction of strain. However, the methods and materials presented in this system are flexible enough to accommodate a number of variations on this theme: strain rate, magnitude, duration, cell type, membrane topography, membrane coating, etc. can all be tailored to the desired application or outcome. This is a robust method for investigating the effects of uniaxial tensile strain applied to cells in vitro.

It is widely understood that mechanical forces in the body can influence cell differentiation and proliferation. Here we present a video protocol demonstrating the use of a custom-built bioreactor for delivering uniaxial cyclic tensile strain to stem cells cultured on flexible micropatterned substrates.

The role of mechanical forces in the development and maintenance of biological tissues is well documented, including several mechanically regulated ...

Biology

Utilizing Transcranial Magnetic Stimulation to Study the Human Neuromuscular System

Transcranial magnetic stimulation (TMS) has been in use for more than 20 years 1, and has grown exponentially in popularity over the past decade. While the use of TMS has expanded to the study of many systems and processes during this time, the original application and perhaps one of the most common uses of TMS involves studying the physiology, plasticity and function of the human neuromuscular system. Single pulse TMS applied to the motor cortex excites pyramidal neurons transsynaptically 2 (Figure 1) and results in a measurable electromyographic response that can be used to study and evaluate the integrity and excitability of the corticospinal tract in humans 3. Additionally, recent advances in magnetic stimulation now allows for partitioning of cortical versus spinal excitability 4,5. For example, paired-pulse TMS can be used to assess intracortical facilitatory and inhibitory properties by combining a conditioning stimulus and a test stimulus at different interstimulus intervals 3,4,6-8. In this video article we will demonstrate the methodological and technical aspects of these techniques. Specifically, we will demonstrate single-pulse and paired-pulse TMS techniques as applied to the flexor carpi radialis (FCR) muscle as well as the erector spinae (ES) musculature. Our laboratory studies the FCR muscle as it is of interest to our research on the effects of wrist-hand cast immobilization on reduced muscle performance6,9, and we study the ES muscles due to these muscles clinical relevance as it relates to low back pain8. With this stated, we should note that TMS has been used to study many muscles of the hand, arm and legs, and should iterate that our demonstrations in the FCR and ES muscle groups are only selected examples of TMS being used to study the human neuromuscular system.

Transcranial magnetic stimulation (TMS) is a non-invasive tool to gain insight on the physiology and function of the human nervous system. Here, we present our TMS techniques to study cortical excitability of the upper limb and lumbar musculature.

Transcranial magnetic stimulation (TMS) has been in use for more than 20 years 1, and has grown exponentially in popularity over the past decade.  While ...

Design of a Biaxial Mechanical Loading Bioreactor for Tissue Engineering

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 &mu;m and a resolution of 20 nm, translating to a positional accuracy of less than 3 &mu;m 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 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  ...

Bioengineering

A Multi-Cue Bioreactor to Evaluate the Inflammatory and Regenerative Capacity of Biomaterials under Flow and Stretch

The use of resorbable biomaterials to induce regeneration directly in the body is an attractive strategy from a translational perspective. Such materials induce an inflammatory response upon implantation, which is the driver of subsequent resorption of the material and the regeneration of new tissue. This strategy, also known as in situ tissue engineering, is pursued to obtain cardiovascular replacements such as tissue-engineered vascular grafts. Both the inflammatory and the regenerative processes are determined by the local biomechanical cues on the scaffold (i.e., stretch and shear stress). Here, we describe in detail the use of a custom-developed bioreactor that uniquely enables the decoupling of stretch and shear stress on a tubular scaffold. This allows for the systematic and standardized evaluation of the inflammatory and regenerative capacity of tubular scaffolds under the influence of well-controlled mechanical loads, which we demonstrate on the basis of a dynamic co-culture experiment using human macrophages and myofibroblasts. The key practical steps in this approach&#8212;the construction and setting up of the bioreactor, preparation of the scaffolds and cell seeding, application and maintenance of stretch and shear flow, and sample harvesting for analysis&#8212;are discussed in detail.

The goal of this protocol is to execute a dynamic co-culture of human macrophages and myofibroblasts in tubular electrospun scaffolds to investigate material-driven tissue regeneration, using a bioreactor which enables the decoupling of shear stress and cyclic stretch.

The use of resorbable biomaterials to induce regeneration directly in the body is an attractive strategy from a translational perspective. Such materials ...

Engineering

3D Magnetic Stem Cell Aggregation and Bioreactor Maturation for Cartilage Regeneration

Cartilage engineering remains a challenge due to the difficulties in creating an in vitro functional implant similar to the native tissue. An approach recently explored for the development of autologous replacements involves the differentiation of stem cells into chondrocytes. To initiate this chondrogenesis, a degree of compaction of the stem cells is required; hence, we demonstrated the feasibility of magnetically condensing cells, both within thick scaffolds and scaffold-free, using miniaturized magnetic field sources as cell attractors. This magnetic approach was also used to guide aggregate fusion and to build scaffold-free, organized, three-dimensional (3D) tissues several millimeters in size. In addition to having an enhanced size, the tissue formed by magnetic-driven fusion presented a significant increase in the expression of collagen II, and a similar trend was observed for aggrecan expression. As the native cartilage was subjected to forces that influenced its 3D structure, dynamic maturation was also performed. A bioreactor that provides mechanical stimuli was used to culture the magnetically seeded scaffolds over a 21-day period. Bioreactor maturation largely improved chondrogenesis into the cellularized scaffolds; the extracellular matrix obtained under these conditions was rich in collagen II and aggrecan. This work outlines the innovative potential of magnetic condensation of labeled stem cells and dynamic maturation in a bioreactor for improved chondrogenic differentiation, both scaffold-free and within polysaccharide scaffolds.

Chondrogenesis from stem cells requires fine tuning the culture conditions. Here, we present a magnetic approach for condensing cells, an essential step to initiate chondrogenesis. In addition, we show that dynamic maturation in a bioreactor applies mechanical stimulation to the cellular constructs and enhances cartilaginous extracellular matrix production.

Cartilage engineering remains a challenge due to the difficulties in creating an in vitro functional implant similar to the native tissue. An approach ...

Simultaneous Electrical and Mechanical Stimulation to Enhance Cells' Cardiomyogenic Potential

Cardiovascular diseases are the leading cause of death in developed countries. Consequently, the demand for effective cardiac cell therapies has motivated researchers in the stem cell and bioengineering fields to develop in vitro high-fidelity human myocardium for both basic research and clinical applications. However, the immature phenotype of cardiac cells is a limitation on obtaining tissues that functionally mimic the adult myocardium, which is mainly characterized by mechanical and electrical signals. Thus, the purpose of this protocol is to prepare and mature the target cell population through electromechanical stimulation, recapitulating physiological parameters. Cardiac tissue engineering is evolving toward more biological approaches, and strategies based on biophysical stimuli, thus, are gaining momentum. The device developed for this purpose is unique and allows individual or simultaneous electrical and mechanical stimulation, carefully characterized and validated. In addition, although the methodology has been optimized for this stimulator and a specific cell population, it can easily be adapted to other devices and cell lines. The results here offer evidence of the increased cardiac commitment of the cell population after electromechanical stimulation. Electromechanically stimulated cells show an increased expression of main cardiac markers, including early, structural, and calcium-regulating genes. This cell conditioning could be useful for further regenerative cell therapy, disease modeling, and high-throughput drug screening.

Here we present a protocol for training a cell population using electrical and mechanical stimuli emulating cardiac physiology. This electromechanical stimulation enhances the cardiomyogenic potential of the treated cells and is a promising strategy for further cell therapy, disease modeling, and drug screening.

Cardiovascular diseases are the leading cause of death in developed countries. Consequently, the demand for effective cardiac cell therapies has motivated ...

Evaluation of Stem Cell Therapies in a Bilateral Patellar Tendon Injury Model in Rats

Regenerative medicine provides novel alternatives to conditions that challenge traditional treatments. The prevalence and morbidity of tendinopathy across species, combined with the limited healing properties of this tissue, have prompted the search for cellular therapies and propelled the development of experimental models to study their efficacy. Umbilical cord matrix-derived mesenchymal stem cells (UCM-MSC) are appealing candidates because they are abundant, easy to collect, circumvent the ethical concerns and risk of teratoma formation, yet resemble primitive embryonic stem cells more closely than adult tissue-derived MSCs. Significant interest has focused on chitosan as a strategy to enhance the properties of MSCs through spheroid formation. This paper details techniques to isolate UCM-MSCs, prepare spheroids on chitosan film, and analyze the effect of spheroid formation on surface marker expression. Consequently, creation of a bilateral patellar tendon injury model in rats is described for in vivo implantation of UCM-MSC spheroids formed on chitosan film. No complication was observed in the study with respect to morbidity, stress rising effects, or tissue infection. The total functional score of the operated rats at 7 days was lower than that of normal rats, but returned to normal within 28 days after surgery. Histological scores of tissue-healing confirmed the presence of a clot in treated defects evaluated at 7 days, absence of foreign body reaction, and progressing healing at 28 days. This bilateral patella tendon defect model controls inter-individual variation via creation of an internal control in each rat, was associated with acceptable morbidity, and allowed detection of differences between untreated tendons and treatments.

This paper describes the preparation and evaluation of umbilical cord matrix-derived mesenchymal stem cells spheroids with a bilateral patellar tendon defect model in a rat. This model was associated with an acceptable morbidity and was found to detect differences between untreated and treated tendons, and between the two treatments tested.

Regenerative medicine provides novel alternatives to conditions that challenge traditional treatments. The prevalence and morbidity of tendinopathy across ...

Harnessing iPSC-Derived iTenocytes for Effective Cell-Based Therapy

Generation of Induced Pluripotent Stem Cell-Derived iTenocytes via Combined Scleraxis Overexpression and 2D Uniaxial Tension

Today&#39;s challenges in tendon and ligament repair necessitate the identification of a suitable and effective candidate for cell-based therapy to promote tendon regeneration. Mesenchymal stromal cells (MSCs) have been explored as a potential tissue engineering strategy for tendon repair. While they are multipotent and have regenerative potential in vivo, they are limited in their self-renewal capacity and exhibit phenotypic heterogeneity. Induced pluripotent stem cells (iPSCs) can circumvent these limitations due to their high self-renewal capacity and unparalleled developmental plasticity. In tenocyte development, Scleraxis (Scx) is a crucial direct molecular regulator of tendon differentiation. Additionally, mechanoregulation has been shown to be a central element guiding embryonic tendon development and healing. As such, we have developed a protocol to encapsulate the synergistic effect of biological and mechanical stimulation that may be essential for generating tenocytes. iPSCs were induced to become mesenchymal stromal cells (iMSCs) and were characterized with classic mesenchymal stromal cell markers via flow cytometry. Next, using a lentiviral vector, the iMSCs were transduced to stably overexpress SCX (iMSCSCX+). These iMSCSCX+ cells can be further matured into iTenocytes via uniaxial tensile loading using a 2D bioreactor. The resulting cells were characterized by observing the upregulation of early and late tendon markers, as well as collagen deposition. This method of generating iTenocytes can be used to assist researchers in developing a potentially unlimited off-the-shelf allogeneic cell source for tendon cell therapy applications.

Author Spotlight: Advancements in Cell and Tissue Engineering for Tendon Repair 

This article describes a procedure to produce iTenocytes by generating iPSC-derived mesenchymal stromal cells with combined overexpression of Scleraxis using a lentiviral vector and uniaxial stretching via a 2D bioreactor.

Today&#39;s challenges in tendon and ligament repair necessitate the identification of a suitable and effective candidate for cell-based therapy to ...

Fabricating Mouse Tendon Assembloids to Study Cellular Interactions in Disease

Engineering Tendon Assembloids to Probe Cellular Crosstalk in Disease and Repair

Tendons enable locomotion by transferring muscle forces to bones. They rely on a tough tendon core comprising collagen fibers and stromal cell populations. This load-bearing core is encompassed, nourished, and repaired by a synovial-like tissue layer comprising the extrinsic tendon compartment. Despite this sophisticated design, tendon injuries are common, and clinical treatment still relies on physiotherapy and surgery. The limitations of available experimental model systems have slowed the development of novel disease-modifying treatments and relapse-preventing clinical regimes. 
In vivo human studies are limited to comparing healthy tendons to end-stage diseased or ruptured tissues sampled during repair surgery and do not allow the longitudinal study of the underlying tendon disease. In vivo animal models also present important limits regarding opaque physiological complexity, the ethical burden on the animals, and large economic costs associated with their use. Further, in vivo animal models are poorly suited to systematic probing of drugs and multicellular, multi-tissue interaction pathways. Simpler in vitro model systems have also fallen short. One major reason is a failure to adequately replicate the three-dimensional mechanical loading necessary to meaningfully study tendon cells and their function. 
The new 3D model system presented here alleviates some of these issues by exploiting murine tail tendon core explants. Importantly, these explants are easily accessible in large numbers from a single mouse, retain 3D in situ loading patterns at the cellular level, and feature an in vivo-like extracellular matrix. In this protocol, step-by-step instructions are given on how to augment tendon core explants with collagen hydrogels laden with muscle-derived endothelial cells, tendon-derived fibroblasts, and bone marrow-derived macrophages to substitute disease- and injury-activated cell populations within the extrinsic tendon compartment. It is demonstrated how the resulting tendon assembloids can be challenged mechanically or through defined microenvironmental stimuli to investigate emerging multicellular crosstalk during disease and injury.

Author Spotlight: Advancing Tendon Research by Developing Mouse Assembloids to Understand Cellular Mechanisms

Here, we present an assembloid model system to mimic tendon cellular crosstalk between the load-bearing tendon core tissue and an extrinsic compartment containing cell populations activated by disease and injury. As an important use case, we demonstrate how the system can be deployed to probe disease-relevant activation of extrinsic endothelial cells.

Tendons enable locomotion by transferring muscle forces to bones. They rely on a tough tendon core comprising collagen fibers and stromal cell ...

Three-Step Organoid Model for Tendon Research and Engineering

Demonstration of Self-Assembled Cell Sheet Culture and Manual Generation of a 3D Tendon/Ligament-Like Organoid by using Human Dermal Fibroblasts

Tendons and ligaments (T/L) are strong hierarchically organized structures uniting&#160;the&#160;musculoskeletal system. These tissues have a strictly arranged collagen type&#160;I-rich extracellular matrix (ECM) and T/L-lineage cells mainly positioned in parallel rows. After injury, T/L require a long time for rehabilitation with high failure risk and often unsatisfactory repair outcomes. Despite recent advancements in T/L biology research, one of the remaining challenges is that the T/L field still lacks a standardized differentiation protocol that is able to recapitulate T/L formation process in vitro. For example, bone and fat differentiation of mesenchymal precursor cells require just standard two-dimensional (2D) cell culture and the addition of specific stimulation media. For differentiation to cartilage, three-dimensional (3D) pellet culture and supplementation of TGF&#223; is necessary. However, cell differentiation to tendon needs a very orderly 3D culture model, which ideally should also be subjectable to dynamic mechanical stimulation. We have established a 3-step (expansion, stimulation, and maturation) organoid model to form a 3D rod-like structure out of a self-assembled cell sheet, which delivers a natural microenvironment with its own ECM, autocrine, and paracrine factors. These rod-like organoids have a multi-layered cellular architecture within rich ECM and can be handled quite easily for exposure to static mechanical strain. Here, we demonstrated the 3-step protocol by using commercially available dermal fibroblasts. We could show that this cell type forms robust and ECM-abundant organoids. The described procedure can be further optimized in terms of culture media and optimized toward dynamic axial mechanical stimulation. In the same way, alternative cell sources can be tested for their potential to form T/L organoids and thus undergo T/L differentiation. In sum, the established 3D T/L organoid approach can be used as a model for tendon basic research and even for scaffold-free T/L engineering.

Author Spotlight: Advancing Tendon Tissue Engineering with 3D Organoid Models

Herein, we demonstrate a three-step organoid model (two-dimensional [2D] expansion, 2D stimulation, three-dimensional [3D] maturation) offering a promising tool for tendon fundamental research and a potential scaffold-free method for tendon tissue engineering.

Tendons and ligaments (T/L) are strong hierarchically organized structures uniting&#160;the&#160;musculoskeletal system. These tissues have a strictly ...

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

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