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,

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 mont....

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