A Protocol to Acquire the Degenerative Tenocyte from Humans

Summary

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.

Abstract

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.

Introduction

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 expectancy^1,2. 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-regulation^3,4,5,6,7,8. 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'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 therapy^10,11,12.

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 pathology^13,14.

Most papers using human samples were analyzed histologically or performed the in vitro experiment based on a healthy human tenocyte instead of a degenerative tenocyte^{15,16,17,18,19,20,21}. 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 human^22,23. 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.

Protocol

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

1. 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.
2. Use the following inclusion criteria: over 18 years of age; a history of lateral epicondylitis for a minimum of six months; recalcitrant for non-operative treatment; at least three months have passed since injection of corticosteroid, platelet rich plasma, or botulinum toxin
3. Exclude the following: pregnant women; patients with a history of cervical spine disease, elbow arthritis, rheumatologic disease, surgery around elbow, and nerve entrapment syndrome; patients refusing to donate tissue.
4. Surgical technique
   ​NOTE: See Figure 1²⁴.
   1. After general anesthesia by isoflurane and endotracheal intubation, put the arm on the table and apply a tourniquet. Then apply betadine agent on the whole arm and drape with an aseptic surgical sheet.
   2. Expose the lateral elbow through a 3–5 cm incision with No. 15 surgical scalpel blade just anteromedial to the lateral epicondyle and proximal to the level of the joint.
   3. Visually identify the extensor carpi radialis longus (ECRL) and extensor aponeurosis interface and make a splitting incision 2–3 mm in depth between the ECRL and extensor aponeurosis with a No. 15 surgical scalpel blade at the identified interface.
   4. Extract the ECRL anteriorly, which will bring the pathologic extensor carpi radialis longus brevis (ECRB) origin below into direct view.
   5. Visually identify the pathologic degenerative tissue by its characteristic dull grayish color and usually edematous and friable tissue.
   6. After identification, carefully resect all degenerative tissue sharply using No. 15 surgical scalpel and mini-blades.
   7. Embed the resected tissue (about 1 cm³) into phosphate buffered saline (PBS) immediately and remove all the surrounding tissue except tendon tissue carefully into about 10 mL of PBS as soon as possible.
   8. In cases with bony exostosis at the lateral epicondyle, remove exostosis using a rongeur and smoothen it with a rasp. Make multiple drill holes (diameter 1.6 mm, about 6–8 holes) in the anterolateral condyle to enhance vascular supply. This is optional.
   9. Close the wound layer by layer using non-absorbable suture material.
5. Healthy control tenocyte
   1. Obtain healthy tenocytes from the remaining tendon of the auto-hamstring or auto-patella tendon during anterior cruciate ligament reconstruction, and use it as the control for the following experiment²⁵.
      NOTE: The number of samples used in this study were three in each group.

2. Histology

1. Store about half of the harvested tissue in neutral buffered 10% formalin in a chemical fume hood or equivalent.
2. Embed the tissue in a paraffin block (4–5 mL) and section it in 4–5 µm sections coronally.
3. Stain sections from control and degenerative tendon with Hematoxylin and Eosin stain (H&E) and Alcian blue stain at a pH of 2.5. Perform H&E staining in an automated stain machine as follows.
   1. Deparaffinize using xylene (3 times, 5 min, respectively).
   2. Hydrate from graded alcohols to water (100% alcohol, 4 min; 100% alcohol, 3 min; 95% alcohol, 2 min; 70% alcohol, 1 min; water, 2 min).
   3. Stain in Hematoxylin for 5 min. Wash well in water for 2 min.
   4. Differentiate in 1% acid alcohol (1% HCl in 70% alcohol) for 3 s. Wash well in water for 3 min.
   5. Neutralize HCl by dipping in an 0.5% ammonia for 10 s, followed by a 3 min water wash.
   6. Stain in Eosion for 5 min.
   7. Dehydrate through alcohols (80% alcohol, 30 s; 95% alcohol, 1 min; 100% alcohol 1 min, 100% alcohol 2 min).
   8. Clear in xylene (3 times, 2 min, 2 min, and 3 min, respectively).
   9. Mount with a cover slide.
4. Perform immunohistochemitry (IHC) using the Bond Polymer Refine Detection kit according to the manufacturer's instructions with minor modifications.
   1. After deparaffinization using xylene, perform an antigen retrieval procedure using Bond Epitope Retrieval Solution for 30 min at 100 °C. The Bond Epitope Retrieval Solution contains a citrate based buffer and surfactant (1 L, pH 5.9–6.1).
   2. Quench endogenous peroxidases by incubating the tissue with hydrogen peroxide for 5 min.
   3. Incubate the sections for 15 min at an ambient temperature (21-22 °C) with primary antibodies against vascular endothelial growth factor (VEGF) (1:200), tenomodulin (1:200), or mohawk (1:200).
   4. Perform secondary antibody tagging with a biotin-free polymeric horseradish peroxidase-linker antibody conjugate system.
      1. Use secondary rabbit anti mouse IgG in 10% (v/v) animal serum in Tris-buffered saline/0.09% 2-Methyl-4-isothiazolin-3-one solution to localize mouse antibodies.
      2. Use polymer anti-rabbit Poly-HRP-IgG (<25 µg/mL) containing 10% (v/v) animal serum in Tris-buffered saline/0.09% to localize rabbit antibodies.
      3. Use 3,3'-Diaminobenzidine tetrahydrochloride hydrate in a stabilizer solution to visualize the complex via a brown precipitate.
      4. Counterstain with <0.1% hematoxylin (blue) to visualize the cell nuclei.
5. Observe the slides stained with H&E, Alcian blue stains, and IHC for VEGF, tenomodulin, and mohawk under light microscope and obtain representative microscopic images of each slide under one high power field (400X).

3. Tenocyte Culture

1. Tissue preparation
   1. Prepare the tendon sample by removing all the surrounding tissue thoroughly with micro-scissors and forceps.
   2. Wash with PBS and mince into small pieces (about 1 mm³ or less).
   3. Digest tissue for 1 h in 30 mL of Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 30 mg/mL Collagenase II at 37 °C.
   4. Pass cells through a 100 µm cell strainer.
   5. Deactivate Collagenase II by adding 1 mL of fetal bovine serum (FBS).
   6. Centrifuge at 196 x g for 5 min.
2. Cell culture
   1. Seed 3 x 10⁵ cells into a 100 mm dish and culture with DMEM supplemented with 10% FBS and 1% Antibiotic-Antimycotic solution at 37 °C in a 5% CO₂ incubator for 1 week.
   2. Change the culture medium every three days until 80% confluence occurs. Culture both healthy control and degenerative cell samples until passage four and use for experiment.

4. Immunocytochemistry (ICC)

1. Transfer 200 µL of cultured cell (2 x 10⁵ cells) to the eight wells of a chamber slide and grow the cells to confluence with the addition of fresh media for 1 day.
2. Wash the cells with PBS and fix with 500 µL of 4% formaldehyde per well.
3. Incubate the slides with 0.5% Triton X-100 in PBS for 10 min for permeabilization of the membranes.
4. Block in PBS with 1% bovine serum albumin and 0.05% TWEEN-20 for mohawk and tenomodulin, respectively.
5. Incubate in the dark with mouse anti-mohawk monoclonal antibody (1:100 dilution) and goat anti-tenomodulin antibody (1:100 dilution) at 4 °C overnight.
6. Incubate the secondary antibodies (Alexa 555-anti goat (1:200) for tenomodulin; Alexa 488-anti mouse (1:200) for mohawk) in the dark for 1 h at room temperature.
7. Mount with 0.5 µL of a fluorescence mounting medium.
8. Analyze using a fluorescence microscope (400X).

5. Proliferation Assay

1. Measure cell viabilities using water-soluble tetrazolium (WST)-1.
   1. Seed both healthy and degenerative cells into 96 well plates at a density of 2 x 10³ cells per well for 24, 72, and 120 h.
   2. After incubation, add 10 µL of the 10% WST-1 solution to each well.
   3. Incubate the plates for 3 h at 37 °C in 5% CO₂.
   4. Measure the optical densities at 450 nm using a microplate reader. Take readings at least three times at all time points for each sample.
2. Measure cellular DNA amount using the BrdU assay.
   1. Seed both healthy and degenerative cell into 96 well plates at a density of 2 x 10³ cells per well for 24, 72, and 120 h.
   2. After incubation, add 10 µL of 10% BrdU solution to each well.
   3. Incubate the plates for 3 h at 37 °C in 5% CO₂.
   4. Add 100 µL/well of the Fixing/Denaturing Solution (60–70% ethanol, 0.1–0.5% sodium hydroxide) and keep the plate at room temperature for 30 min.
   5. Add 100 µL/well prepared 1x detection antibody solution and keep the plate at room temperature for 1 h.
   6. Remove solution and wash plate three times with 1x Wash Buffer.
   7. Add 100 µL/well prepared 1x HRP-conjugated secondary antibody solution and keep the plate at room temperature for 30 min.
   8. Remove the solution and wash plate three times with 1x Wash Buffer.
   9. Add 100 µL of 50% TMB Substrate.
   10. Incubate for 30 min at room temperature.
   11. Add 100 µL of STOP Solution.
   12. Measure the optical densities at 450 nm using a microplate reader.
       NOTE: Take readings at least three times at all time points for each sample.

6. Statistical Analysis

1. Collect and analyze data using SPSS.
2. Depict data by mean ± standard error of mean.
3. Use Mann Whitney U test.
4. Consider differences statistically significant when the p value is less than 0.05 (p <0.05).

Results

Histological analyses revealed that the harvested tissue from lateral epicondylitis had the characteristics of a tendinopathic tendon.H&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...

Discussion

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 more^26,27. 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...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Scalpel	Kisanbio	KS-Q0306-15	No. 15
Mini-blade	Beaver	374769
Dulbecco's modified Eagle's medium (DMEM)	Gibco	11995065
PBS	Gibco	14190250
fetal bovine serum (FBS)	Gibco	16000044
50 mM ascorbic acid-2-phosphate	Sigma-Aldrich	A5960
Antibiotic-Antimycotic solution	Gibco	15240062
4% formaldehyde	Bio-solution	BP031
Triton X-100	Sigma-Aldrich	X100-100ml
BSA	Rdtech	C0082
TWEEN 20	Sigma-Aldrich	P9416-100ml
MKX (C-5)	Santa cruz biotechnology	sc-515878
Tenomodulin (N-14)	Santa cruz biotechnology	sc-49325
Fluorescence Mounting Medium	DAKO	S3023
DAPI (4',6-Diamidino-2-Phenylindole, Dihydrochloride)	Thermo Fisher Scientific	D1306
WST-1	Dojindo Molecular Technologies	CK04
BrdU Cell Proliferation Assay Kit	Cell Signaling Technology	#6813
TRIzol Reagent	Invitrogen	15596018
iScript cDNA Synthesis Kit	Bio-Rad	170-8891
TaqMan Gene Expression Master Mix	Applied Biosystems	4369016
GAPDH	Thermo Fisher Scientific	Hs02786624_g1
COL3A1	Thermo Fisher Scientific	Hs00943809_m1
ACTA2	Thermo Fisher Scientific	Hs00426835_g1
TAC1	Thermo Fisher Scientific	Hs00243225_m1
TACR1	Thermo Fisher Scientific	Hs00185530_m1
PTGS2	Thermo Fisher Scientific	Hs00153133_m1
ACTB	Thermo Fisher Scientific	Hs99999903_m1
Cell Strainers (100 µm)	Corning	352360
100mm culture dish	Thermo Fisher Scientific	8188207
8-well Chamber Slide	Thermo Fisher Scientific	154534
96 Well Clear Flat Bottom Polystyrene TC-Treated Microplates	Corning	3596
Nikon Eclipse 50i Microscope	Nikon
VERSA max microplate reader	Molecular Devices
CFX96 Real-Time PCR Detection System	Bio-Rad
Formalin solution, neutral buffered, 10%	Sigma-Aldrich	HT501128
Paraffins	Leica Biosystems	3801340
Ethanol	JUNSEI CHEMICAL	90303-2185
Hematoxylin	DAKO	CS70030-2
Eosin	DAKO	CS70130-2
Alcian blue	DAKO	AR16011-2
Citric acid	Sigma-Aldrich	251275
Xylene	JUNSEI CHEMICAL	25165-0430
Endogenous peroxidases	DAKO	S200380-2
Canada balsam	JUNSEI CHEMICAL	23255-1210
Microtome Blade	FEATHER	A35
Slide glass	SUPERIOR	1000612
Cover glass	Marienfeld-Superior	101050
VEGF	Santa cruz biotechnology	sc-7269
SPSS Software	IBM		Ver. 18.0
Multi-purpose Centrifuge	LABOGENE	1248R