Name: a protocol to acquire the degenerative tenocyte from humans
Uploaded: 2018-06-09T14:30:00
Description: Watch this Scientific Journal Video about A Protocol to Acquire the Degenerative Tenocyte from Humans at JoVE.com

This research was supported by a grant from the Korea Health Technology R&#38;D Project through the Korea Health Industry Development Institute (KHIDI), which was funded by the Ministry of Health &#38; Welfare, Republic of Korea (grant number: HI16C1559).

The protocol was conducted in accordance with the Declaration of Helsinki and the protocol was approved by the Institutional Review Board at CHA Bundang Medical Center.
1. Degenerative Tendon Tissue Harvest from the Patient
<ol>
	<li>Diagnose lateral epicondylitis by taking a medical history and using physical exam findings: tenderness over the tendinous origin close to the lateral epicondyle; pain evoked by resisted extension of the wrist joint.</li>
	<li>Use the following inclusion criteri...

<ol>
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V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mesenchymal+stem+cells+and+insulin-like+growth+factor-I+gene-enhanced+mesenchymal+stem+cells+improve+structural+aspects+of+healing+in+equine+flexor+digitorum+superficialis+tendons.">Mesenchymal stem cells and insulin-like growth factor-I gene-enhanced mesenchymal stem cells improve structural aspects of healing in equine flexor digitorum superficialis tendons.</a> Journal of Orthopedic Research. 27 (10), 1392-1398 (2009).</li><li>Durgam, S. S., Stewart, A. A., Sivaguru, M., Wagoner Johnson, A. J., Stewart, M. 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A number of previous studies have reported how to create chronic tendinopathic animal models using different procedures such as collagenase or kartogenin injection, treadmill running, and more26,27. Although numerous studies showed promising therapeutic effects based on these animal models, experiments using the human degenerative tenocyte would be crucial in the field of tendinopathy in order to reproduce the efficacy of treatment. In this article, we establishe...

Tendinopathy is a chronic degenerative musculoskeletal condition that develops in various parts of the body. Recently, the number of cases of tendinopathy has increased greatly in the developed world due to growing participation in recreational sports and increased life expectancy1,2. The cause of tendinopathy is considered to be multifactorial and these causes include ischemia, oxygen free radical injuries, an imbalance between vasoconstrictor and vasodilator innervations, internal micro-tears, and changes to neuro-regulation3,4,5,6,7,8. Most treatments for tendinopathy only relieve its symptoms. Moreover, treatments without tissue regeneration require a long time for rehabilitation and achieve a limited response from injured tendons, which imposes a clinical challenge for physicians9.
The incompetence of current treatment options along with the lack of the degenerative tendon&#39;s ability to self-heal has lead researchers to take interest in exploring alternative treatment strategies. Recently, new studies reported many promising results for enhancing the healing efficacy of the tendinopathy tendons using growth factors, stem cell based therapy, and gene therapy10,11,12.
Through a literature review, we found that the involved studies may be divided into two categories based on their analysis materials: animal models such as a rat, a mouse, or a rabbit; and human models. With regard to the animal model, currently there are two popular techniques to generate tendinopathy: chemical induction of injury or mechanical overloading the model. However, each animal model was limited in reproducing the complex human tendinopathy pathology13,14.
Most papers using human samples were analyzed histologically or performed the in vitro experiment based on a healthy human tenocyte instead of a degenerative tenocyte15,16,17,18,19,20,21. Only a few papers reported that they used a human degenerative tenocyte, but they did not describe in detail the protocol used to get the degenerative tenocyte from the human22,23. In this context, it should be noted that successful results from either the animal model or the healthy tissue/tenocyte may not necessarily predict human efficacy or efficacious dosing because tendon degeneration is a complicated process and the pathogenesis is still not fully understood.
Collectively, it is necessary to describe the standard protocol for obtaining the degenerative tenocyte from human tissue without causing adverse effects to the donor. This article describes a protocol on how to acquire the human degenerative tenocyte. To validate the protocol, the harvested tissues were analyzed histologically. Then, the cultured cell was confirmed as the degenerative tenocyte by using immunocytochemistry (ICC), a quantitative real time-polymerase chain reaction (qRT-PCR), and a viability assay.

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			<td height="21" style="height:21px;width:235px;">Scalpel</td>
			<td style="width:199px;">Kisanbio</td>
			<td style="width:119px;">KS-Q0306-15</td>
			<td style="width:167px;">No. 15</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">Mini-blade</td>
			<td style="width:199px;">Beaver</td>
			<td style="width:119px;">374769</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:235px;">Dulbecco&#39;s modified Eagle&#39;s medium (DMEM)</td>
			<td style="width:199px;">Gibco</td>
			<td style="width:119px;">11995065</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">Collagenase &#8545;</td>
			<td style="width:199px;">Sigma-Aldrich</td>
			<td style="width:119px;">C6885</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">PBS</td>
			<td>Gibco</td>
			<td style="width:119px;">14190250</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">fetal bovine serum (FBS)</td>
			<td>Gibco</td>
			<td style="width:119px;">16000044</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">50 mM ascorbic acid-2-phosphate</td>
			<td style="width:199px;">Sigma-Aldrich</td>
			<td style="width:119px;">A5960</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">Antibiotic-Antimycotic solution</td>
			<td>Gibco</td>
			<td style="width:119px;">15240062</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">4% formaldehyde</td>
			<td style="width:199px;">Bio-solution</td>
			<td style="width:119px;">BP031</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">Triton X-100&#160;</td>
			<td style="width:199px;">Sigma-Aldrich</td>
			<td style="width:119px;">X100-100ml</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">BSA</td>
			<td style="width:199px;">Rdtech</td>
			<td style="width:119px;">C0082</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">TWEEN 20</td>
			<td style="width:199px;">Sigma-Aldrich</td>
			<td style="width:119px;">P9416-100ml</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">MKX (C-5)</td>
			<td style="width:199px;">Santa cruz biotechnology</td>
			<td style="width:119px;">sc-515878</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">Tenomodulin (N-14)</td>
			<td style="width:199px;">Santa cruz biotechnology</td>
			<td style="width:119px;">sc-49325</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">Fluorescence Mounting Medium</td>
			<td style="width:199px;">DAKO</td>
			<td style="width:119px;">S3023</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:235px;">DAPI (4&#39;,6-Diamidino-2-Phenylindole, Dihydrochloride)</td>
			<td style="width:199px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">D1306</td>
			<td></td>
		</tr>
		<tr height="46">
			<td height="46" style="height:46px;width:235px;">WST-1</td>
			<td style="width:199px;">Dojindo Molecular Technologies</td>
			<td style="width:119px;">CK04</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">BrdU Cell Proliferation Assay Kit</td>
			<td style="width:199px;">Cell Signaling Technology</td>
			<td style="width:119px;">#6813</td>
			<td></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:235px;">TRIzol Reagent</td>
			<td style="width:199px;">Invitrogen</td>
			<td style="width:119px;">15596018</td>
			<td></td>
		</tr>
		<tr height="23">
			<td height="23" style="height:23px;width:235px;">iScript cDNA Synthesis Kit&#160;</td>
			<td style="width:199px;">Bio-Rad</td>
			<td style="width:119px;">170-8891</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:235px;">TaqMan Gene Expression Master Mix</td>
			<td style="width:199px;">Applied Biosystems</td>
			<td style="width:119px;">4369016</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">GAPDH</td>
			<td style="width:199px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">Hs02786624_g1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">COL3A1</td>
			<td style="width:199px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">Hs00943809_m1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">ACTA2</td>
			<td style="width:199px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">Hs00426835_g1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">TAC1</td>
			<td style="width:199px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">Hs00243225_m1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">TACR1</td>
			<td style="width:199px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">Hs00185530_m1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">PTGS2</td>
			<td style="width:199px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">Hs00153133_m1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">ACTB</td>
			<td style="width:199px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">Hs99999903_m1</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">Cell Strainers (100 &#181;m)&#160;</td>
			<td style="width:199px;">Corning</td>
			<td style="width:119px;">352360</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">100mm culture dish</td>
			<td style="width:199px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">8188207</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:235px;">8-well Chamber Slide</td>
			<td style="width:199px;">Thermo Fisher Scientific</td>
			<td style="width:119px;">154534</td>
			<td></td>
		</tr>
		<tr height="63">
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			<td style="width:199px;">IBM</td>
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			<td style="width:199px;">LABOGENE</td>
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</table>

a protocol to acquire the degenerative tenocyte from humans

Tendinopathy, a painful condition that develops in response to tendon degeneration, is on the rise in the developed world due to increasing physical activity and longer life expectancy. Despite its increasing prevalence, the underlying pathogenesis still remains unclear, and treatment is generally symptomatic. Recently, numerous therapeutic options, including growth factors, stem cells, and gene therapy, were investigated in hopes of enhancing the healing potency of the degenerative tendon. However, the majority of these research studies were conducted only on animal models or healthy human tenocytes. Despite some studies using pathological tenocytes, to the best of our knowledge there is currently no protocol describing how to obtain human degenerative tenocytes. The aim of this study is to describe a standard protocol for acquiring human degenerative tenocytes. Initially, the tendon tissue was harvested from a patient with lateral epicondylitis during surgery. Then biopsy samples were taken from the extensor carpi radialis brevis tendon corresponding to structural changes observed at the time of surgery. All of the harvested tendons appeared to be dull, gray, friable, and edematous, which made them visually distinct from the healthy ones. Tenocytes were cultured and used for experiments. Meanwhile, half of the harvested tissues were analyzed histologically, and it was shown that they shared the same key features of tendinopathy (angiofibroblastic dysplasia or hyperplasia). A secondary analysis by immunocytochemistry confirmed that the cultured cells were tenocytes with the majority of the cells having positive stains for mohawk and tenomodulin proteins. The qualities of the degenerative nature of tenocytes were then determined by comparing the cells with the healthy control using a proliferation assay or qRT-PCR. The degenerative tenocyte displayed a higher proliferation rate and similar gene expression patterns of tendinopathy that matched previous reports. Overall, this new protocol might provide a useful tool for future studies of tendinopathy.

Histological analyses revealed that the harvested tissue from lateral epicondylitis had the characteristics of a tendinopathic tendon.H&#38;E section of tendinopathy degenerative tendon revealed a disorganized collagen bundle with a loss of polarity and fine straight, strongly packed parallel fiber structures. Histological signs suggestive of degeneration such as higher cellularity and enlarged nuclei without the typical spindle shape were common in samples. Additionally, collagen bundles...

Watch this Scientific Journal Video about A Protocol to Acquire the Degenerative Tenocyte from Humans at JoVE.com

A Protocol to Acquire the Degenerative Tenocyte from Humans

In vitro use of degenerative tenocytes is essential when investigating the efficacy of novel treatment on tendinopathy. However, most research studies use only the animal model or a healthy tenocyte. We propose the following protocol to isolate human degenerative tenocytes during surgery.

Tendinopathy, a painful condition that develops in response to tendon degeneration, is on the rise in the developed world due to increasing physical ...

This method can help answer key questions about human degenerative tenocyte biology as only few studies have described human degenerative tenocyte isolation and culture in detail. The main advantage of this technique is that we can obtain human degenerative tenocyte without additional patient injury because the degenerative tissue is acquired during standard epicondylitis surgery. Demonstrating the procedure will be Dr.Soo-Hong Han, Minjung Baek, and Hyung Kyung Kim from my Level Three.To acquire a degenerative tendocyte tissue sample, use a number 15 surgical scalpel to make a three to five centimeter skin incision just anterior medial to the lateral epicondyle, and proximal to the level of the joint to expose the lateral elbow and visually locate the extensor carpi radialis longus in the extensor aponeurosis interface. Use the scalpel to make a splitting incision two to three millimeters in depth between the extensor carpi radialis longus and the extensor aponeurosis at the interface. After anterior retraction of the extensor carpi radialis longus, locate the pathologic degenerative tissue by its characteristic dull grayish color and typically edematous and friable tissue, and use the scalpel and mini blades to carefully, but sharply, resect all of the degenerative tissue.When all the tissue has been harvested, close the wound with nonabsorbable suture material according to standard protocols and carefully remove all of the surrounding tissue from an approximately one cubic centimeter piece of tissue sample, placing the clean tendon tissue into 10 milliliters of PBS as soon as it has been trimmed. Then fix about half of the harvested tissue in neutral buffered 10%formalin in a chemical fume hood for 12 hours. To set up a tenocyte culture, transfer the other half of the harvested control and degenerated tissue samples into individual tissue culture dishes containing fresh PBS and mince the tissues into one cubic meter or smaller pieces, digest the tissue fragments in 30 milliliters of DMEM supplemented with 30 milligrams per milliliter of collagenase two per tissue group at 37 degrees Celsius for one hour.At the end of the digestion, filter the cell solutions through individual 100 micron cell strainers and deactivate the collagenase with one milliliter of FBS per tube. Collect the tenocytes by centrifugation and re-suspend the pellets at three times 10 to the fifth cells per 10 milliliters of DMEM supplemented with 10%FBS and 1%antibiotic, antimycotic solution concentration. Then, seed the cells into one 100 centimeter culture dish per condition, and culture the tenocytes in a 37 degree Celsius and 5%carbon dioxide incubator for one week, changing the culture medium every three days until an 80%confluence has been reached.For Immunocytochemical analysis, transfer two times 10 to the fifth cells in 200 microliters of fresh medium to each well of one eight-well chamber slide per experimental group and grow the cells to confluence for 24 hours. The next day, wash the cells with PBS, followed by fixation with 500 microliters of 4%formaldehyde per well, and permeabilization with 0.5%triton X 100 in PBS for 10 minutes. To block any non-specific binding, incubate the cells with 1%bovine serum albumin, and 0.05%Tween 20 in PBS, as experimentally appropriate, followed by incubation with the flourescently-tagged primary antibodies of interest at four degrees Celsius overnight protected from light.The next morning, label the cells with the appropriate secondary antibodies for one hour at room temperature protected from light, and mount the samples with 0.5 microliters of an appropriate fluorescence mounting medium per chamber. Then image the samples on a fluorescence microscope under a 400 times objective. For pathological analysis of the tissue, embed the formal and fixed control and degenerated tendon tissue samples in four to five milliliters of paraffin and acquire four to five micron sections from each paraffin block.For hematoxylin in eosin, or H&E staining, deparaffinize the samples with three five-minute immersions in xylene followed by hydration with graded ethanol immersions and a two-minute tap water wash. Next, label the samples with hematoxylin for five minutes, followed by another two-minute water wash. Dip the rinsed samples in 1%hydrochloric acid and 70%ethanol for three seconds, followed by a three-minute water wash.Neutralize the acid with a 10 second dip in 0.5%ammonia, followed by another three-minute water wash. Label the neutralized samples with the eosin for five minutes, and dehydrate the H&E stained samples with graded ethanol immersions. After three xylene immersions, mount the samples with an appropriate mounting medium and a cover slide.To perform immunohistochemical analysis of the tissue samples, use the bond polymer refined detection kit according to the manufacturer's instructions with the following minor modifications. After deparaffinization with xylene as demonstrated, immerse the samples in bond epitope retrieval solution for 30 minutes at 100 degrees Celsius for antigen retrieval, followed by a five-minute incubation in hydrogen peroxide to quench any endogenous peroxidase. Next, label the sections for fifteen minutes at room temperature with the primary antibodies of interest.At the end of the incubation, label the samples with the appropriate biotin-free polymeric horseradish peroxidase linker secondary antibody conjugate system for 50 minutes at room temperature. Then mount the sections with a cover slip in the appropriate mounting medium and image the samples on a light microscope at a 400 times magnification. H&E analyses reveals that tissue harvested from the degenerated lateral epicondylitis demonstrates disorganized collagen bundles with a loss of polarity and fine, straight, strongly packed parallel fiber structures.Indeed, the collagen bundles are positive for Alcian Blue staining in the degenerated tissue sections indicating an increased proteoglycan and glycosaminoglycan presence within the damaged tissue. Immunohistochemical analysis of the degenerated tendons also indicates a cytoplasmic granular positive reaction to vascular endothelial growth factor that is not observed in control, healthy tendon tissue samples. Both types of cultured tissue sample cells can be confirmed as tenocytes by immunofluorachemistry with most of the cultured cells expressing representative tenocyte markers such as the mohawk and tenomodulin proteins and demonstrating an elongated appearance.Of note, the degenerative tenocytes also demonstrate a higher rate of proliferation in culture compared to tenocytes harvested from healthy controls as well as a higher expression of multiple genes known to be related to the development of tendinopathy. In conclusion, this validated protocol using degenerative tenocyte paves the way for future investigations in tendon biology and for testing new interventions.

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