B.M. Al Matour1, P. Loughran1, K. Nickl1, J. Mulla2, R. Mcconnell1, G. Hussey2, M.J. Scott1 1University Of Pittsburg, Surgery, Pittsburgh, PA, USA 2university of pittsburgh, Pathology, Pittsburgh, PA, USA
Introduction:
Matrix-Bound NanoVesicles (MBV) are nanometer-sized vesicles ranging from 50 to 300nm in size; they’re embedded within the collagen and elastin fibrillar meshwork of the Extracellular Matrix (ECM). Within them, they carry a biologically active cargo (protein, miRNA, and lipids) that have been shown to aid in ECM repair and homeostasis. We hypothesized that tenocytes, key fibroblastic cells, involved partially in ECM production would produce MBV during ECM formation. We also showed in our previous work that MBV express Lysyl Oxidase on their surface, a key enzyme in collagen crosslinking, stressing on their role in ECM maintenance, structural integrity, and homeostasis.
Methods:
Tenocytes were isolated from Sprague-Dawley rat-tail tendons. Briefly, tendons were dissected and teased from their sheaths, rinsed in %1 Penicillin-Streptomycin in PBS. Tendons were then digested in high glucose DMEM containing 20mM Tris (pH = 7.5), 150mM NaCl2, and 3mg/ml collagenase and left on an orbital shaker in an incubator at 37°C until fully homogenized. Digested tissues were filtered through a 70μm cell strainer before being seeded in a 6-well plate and allowed to reach confluency, forming a visible monolayer over a duration of 21 days with media (DMEM, %20 fetal bovine serum, %1 penicillin-streptomycin) change every 3 days.
Samples were fixed in 2.5% glutaraldehyde; they were rinsed, fixed in 1% osmium tetroxide with 1% potassium ferricyanide, dehydrated through a graded series of ethanol and embedded in Poly/Bed® 812 (Luft formulations). Semi-thin (300 nm) sections were cut on a Leica Reichart Ultracut, stained with 0.5% Toluidine Blue in 1% sodium borate and examined under the light microscope. Ultrathin sections (65 nm) were stained with uranyl acetate and Reynold’slead citrate and examined on JEOL 1400 Plus Transmission Electron Microscope (TEM) with a side mount AMT 2k digital camera (Advanced Microscopy Techniques, Danvers, MA).
Results:
TEM images (fig.1) revealed an extracellular population of vesicles embedded within the ECM space. The morphology, location and origin of these vesicle is consistent with previously described MBV, validating our previously published data.
Conclusion:
Unstimulated tenocytes produced MBV as part of their normal physiological growth and proliferation, and parralel to ECM production. The presence of MBV within ECM highlights their integral role in maintaining its integrity and homeostasis, suggesting potential benefits of MBV as adjuvant therapy.