Biological Effect Of Exogenous Prostaglandin E2on Tenocytes In Vitro And Tendon In Vivo

Background and Objective:Tendinopathy is a common disease of sports medicine. It was broadly believed thattendon repetitive overuse caused by various improper sports leads to micro tendon injuryand causes tendon gradual degeneration. Nevertheless, its pathogenesis is still not very clear.Relative studies have found that Prostaglandin E2(PGE2) produced by tenocytes under thecircumstance of stretch loading in vitro significantly increases, that PGE2around tendonsheath also significantly increases after excessive exercises and that overloading tenocytesand PGE2may play an important role in the development of tendinopathy.Normal tendon is rich in collagen, with the majority of type I and a small amount oftype III collagen. As far as tendinopathy is concerned, collagen matrix is changed, and thediscontinuity of collagen fibers is suspended, the structure loosed and fissured. Type IIIcollagen increases for degenerated tendon and achilles tendon of strong load. According theresearch on tendinopathy of the animal model in vivo, it is found that the total amount ofcollagen significantly decreases and type III collagen increases; while in vitro model,tendon proliferation ability decreases, type I collagen decreases and I/III was inverted alongwith the increase of tenocytes passage numbers. Among various factors causing tendoninjury, tendon collagen matrix was remodeled, collagen types transformed and collagenmatrix gradually deposited and degenerated.It still lacks for direct evidence(s) verifying whether PGE2can lead to tendon collagen,extracellular collagen matrix and biomechanical changes. Therefore, this study starts withclinical pathology of tendinopathy, observes tendon pathological properties, analyzesinfluence of exogenous PGE2on tendon cell proliferation and collagen type I, III secretion,explores impacts of exogenous PGE2on tendon collagen types, content and biomechanics,etc., and discuss the relationship between PGE2and tendinopathy.Methods: 1. Histopathological observation of human tendinopathy tissueWe collected obsolete tendon tissues from pathological tendon tendinopathy surgeriesand normal tendon tissue due to trauma or amputation surgeries. Through H-E staining,tenocytes structure and tendon matrix morphology are observed; Through Picric acid-Sirius red staining, tendon collagen matrix changes are observed under polarized lightmicroscopy and changes of collagen content and types are also analyzed.2. The effects of different doses of exogenous PGE2on tenocytes proliferation in vitroand extracellular matrix collagenThe tenocytes were isolated by the means of enzymic-digestion, morphology of thecells were assayed with inverted microscope and collagen type ⅠⅢ of the tenocytes werecharactered by immunofluorescence stain and confocal laser scanning microscope.[~3H]-TdR incorporation by scintillation counting method to measure the tendon cell proliferation,application of different doses of type III collagenase linked immunoassay PGE2role intendon cell culture medium I and type III collagen secretion.3. The effects of exogenous PGE2on collagen content, proportion and biomechanicalcharacteristics of rabbits Achilles tendon in vivoJapanese rabbits, weighing2.0-2.5kg and male or female, were equally randomizedinto two groups: injected with low dose PGE2(50ng) and high dose PGE2(500ng); andone side handlimb was randomly selected to be injected with PGE2in the midsubstance ofthe achilles tendon, another side was injected with same dose saline in the same site. Theinjection was repeated once a week for4weeks or8weeks, and the rabbits were sacrificed1week after the last injection. The Achilles tendons were harvested, paraffin-embedded andexamined using hematoxylin and eosin staining and picric acid-sirius red staining.Polarization microscope was used to observe the distribution and types of collagen fibersand transmission electron microscopy was used to examine the density of the unit area anddiameter of collagen fibers. The changes of amount and distribution of type I collagen andtype III collagen were analyzed with SPSS17.0software. Computer controlled electronicuniversal testing machine was used to observe maximum load and tensile strength of theAchilles tendon. The changes of maximum load and tensile strength were analyzed withSPSS17.0software.Results: 1. Compared with the normal tendon, HE staining showed that collagen fiber of tendonwas disorganized and the structure damaged. Picric acid-sirius red staining showed that thecontent of type I collagen reduced while the content of type III collagen increased, and thewhole collagen matrix was disorganized.2. The tenocytes were isolated successfully by the means of both methods, and grewwell after passage. It can promote tenocytes proliferation in low dose PGE2groups after48hours(P<0.05), while it did not significantly affect tenocytes proliferation after24hours or12hours. It can inhibit tenocytes proliferation in high dose groups(P<0.05). ELISA resultsshowed that the secretion of collagen III was higher in different dose groups than controlgroup (P<0.05)while the secretion of collagen did not significantly change, and the collagenration of type I/type III was inverted(P<0.05).3. HE staining showed that collagen structural damage was observed in low dose andhigh dose groups. Picric acid-sirius red staining showed that the content of type I collagenreduced while the content of type III collagen increased in experimental sides of two groupsafter4weeks and8weeks(P<0.05); In experimental sides,the content of type I collagenwas lower while the content of type III collagen was higher in high dose group than those oflow dose group, and the collagen ration of type I/type III was inverted(P<0.05). By TEM,in experimental sides of two groups after4weeks and8weeks,the unit density of collagenfibers was lower and its diameter was thinner than those of the control sides(P<0.05).Especially the high dose group exhibited remarkable above-mentioned changes(P<0.05).Conclusions:1. Histopathological observation of tendinopathy finds that human tendon collagenmatrix has changed significantly: collagen matrix degeneration, fiber structural damage.The type of collagen changes: Type I collagen reduced while the type III collagen increased.2. The effects of exogenous PGE2on tenocytes proliferation and secretion of typeⅠand type III collagen changed. It can promote tenocytes proliferation or it did notsignificantly affect tenocytes proliferation in low dose PGE2, while it can inhibit tenocytesproliferation in high dose groups. The secretion of collagen III was higher in different dosegroups than control group and the collagen ration of type I/type III was inverted.3. Repetitive exposure of the tendon to PGE2can cause the decrease of type I collagen,the increase of type III collagen, the reverse ratio of type I to type III, reduced unit density of collagen fibers, thinner collagen fibers diameter, and reduced biomechanical properties,according with tendinopathy histopathological features.