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.
