Role And Mechanism Of Calcium Overloading Or Cytoskeleton Injury Caused By Overloading In Tendinopathy

BackgroundTendinopathy, not only an occupational disease, but also a most common sports injurydisease with high incidence, is one of the main diseases in Orthopaedics and SportsMedicine. A wealth of research has showed that minor injury to tendon and immature repaircaused by overloading constitute the direct reason for tendinopathy, with such pathologicalmanifestation as tendon degeneration, extracellular matrix increasing, collagen disorder,irregular hyperplasia of tendon vessel, calcification and ossification of tendon, tendoninsertion labial hyperplasia etc. As is known, various human sports depend on the musclecontraction of tendon or ligament causing bone or joint movements. Various dynamicmechanical stimulations imposed upon the tendon causing remodeling of tendon structuresfinally leads to tendon physiological or pathological change. In the course of transmissionand transformation of mechanical stimulation, all kinds of signal factors play significantroles, with calcium ion as the second messenger, transforming mechanical signal intobio-chemical signal in the cells so as to cause a series of physiological or pathologicalprocesses. In the meantime, cytoskeleton is also an important structure for mechanicaltransmission and signal transduction. Therefore, research on the action and mechanism ofcalcium ion in tendon cells and cytoskeleton in response to mechanical stimulation has beenthe hot issue in current Sports Medicine and Orthopaedics.We hypothesized that mechanic overloading may cause cytoskeleton damage andcalcium overloading in the tenocytes. And mechanical overloading may inducecytoskeleton damage, activate the calcium channel on the cell membrane, induceextracellular calcium influx and intracellular calcium consistency increasing which leads tointracellular calcium overloading, and finally results in intracellular calcium deposits observed in tendons and tendon calcification.In our preliminary phase of research, we have managed to isolate human tenocytes andestablish tenocytes force model in vitro. This endeavor, by applying tenocytes force modelin vitro, administers different calcium inhabitors or exogenous calcium underimmunefluorescence technology and laser confocal microscope with or without mechanicaloverloading, as well as observed cytoskeleton F-actin changes. Observed mechanicalstretch damaged F-actin and increased intracellular calcium concentration, to providetheoretical basis for the pathogenesis of tendinopathy.ObjectiveTo simulate the way of parallel arrangement and stress distribution of tenocytes invivo，observe the intracellular calcium concentration and cytoskeleton damage of tenocytesby mechanical overloading with cyclic mechanical stretch in vitro model of tenocytes andimmunefluorescence technology and laser confocal microscope, discuss the role of calciumoverloading and F-actin damaged in mechanical overloading caused tendinopathy，andmechanism of calcium overloading in tenocytes, clarify possible pathogenesis of calcifictendinopathy and to provide a new approach to prevent and treatment calcific tendinopathy.MethodsⅠ Isolation, culture and identification as well as mechanical stretch effectivenessevaluation of human tenocytes1.Human tenocytes isolation and primary culture from fresh tendon tissue of humansurgical wastes，identification tenocytes phenotype by observed typeⅠcollagen，type Ⅲcollagen and vimentin with immunofluorescence and laser scanning confocal microscope.2. Observed effectiveness mechanical stretching of human tenocytes. Human tenocyteswere stretched at4%,8%,12%magnitude cyclic mechanical stretching with duration of12hours，frequency of1.0Hz,Under the same condition, set the blank the control group,unstretched. Laser scanning confocal microscope and microscope was used to examine thechanges of cell shape，Whether there is amotic and float and arrangement way on themicrogroove silicone membrane stretch system comparison with stretch and unstretchedgroup.Ⅱ Inducing human tendon cells in vitro calcium overloading by various stretchloading and its mechanism 1. Intracellular Calcium Immunofluorescence Analysis.We examined the changes of calcium concentration at0.5Hz under different stretchingconditions. Cells were stretched at4%、8%、12%magnitude and4、8、12h respectively,no-stretching cells were used as blank control unless indicated. After stretch, cells wereImmunofluorescence stained by Fluo-3，photographed and recorded with the ZEISS confocallaser scanning microscopy under20times the objective to selection from eight to tenaperture. One hundred cells of each group were randomly selected and the optical densitywas measured double blindly to indication Intracellular calcium.2. Influence of Extracelluar Calcium Blocking by EGTA，and Calcium overloading bydifferent Calcium Channel inhibitor.Human tenocytes were cultured on the microgroove silicone membrane for24h, thenthe medium was replaced with3ml different concentration （0.5mmol/L or1mmol/L） ofethylene glycol tetraacetic acid, different concentration of MnCl₂（0,0.5mol/L or1.0mmol/L） or Nifedipine （0,20, and40μM）, different concentration of heparin（50-or100ug/L） or Phenothiazine(（20,40or80μM）, cells were stretched at8%magnitude,0.5Hzfor4h, no-stretching cells were used as blank control unless indicated. After stretch, cellswere Immunofluorescence stained by Fluo-3，photographed and recorded with the ZEISSconfocal laser scanning microscopy under20times the objective to selection from eight toten aperture. Fifty cells of each group were randomly selected and the optical density wasmeasured double blindly to indication Intracellular calcium.3. Mechanical stretch can activate calcium channel opening and induce calcium influxon tenocytes membrane.Difference concentration of CaCl₂（3mmol/L、5mmol/L and10mmol/L） was prepared.Tenocytes were cultured on the microgroove silicone membrane for24h, the medium wasremoved and cells were washed twice with PBS. In group one, cells were only incubated4hwith3mmol/L、5mmol/L or10mmol/L calcium, respectively; in group two, cells werestretched at8%magnitude,0.5Hz for4h under treatment of3mmol/L、5mmol/L or10mmol/L calcium, respectively. After stretch, calcium immunofluorescence was carriedout according to section1.Ⅲ Influence of various stretch loading and calcium overloading on tendoncytoskeleton 1. Cycle Mechanical stretch can be induced F-actin damage of human tenocytes.Human tenocytes were cultured on the microgroove silicone membrane for24h，stretched under4%,8%,12%cyclic mechanical stretching with duration of2,4,8,12,24hours, frequency of0.5Hz and1.0H. Laser scanning confocal microscope was used toexamine the changes of F-actin and nucleus after immunofluorescent staining at differentcyclic mechanical stretching on human tenocyte. The single cell average fluorescenceintensity was measured with image-analysis system in the photos of human tenocytecytoskeleton.2. Cytoskeleton injury by calcium overloadingDifference concentration of CaCl₂（3mmol/L、5mmol/L and10mmol/L） was prepared.the medium was removed and the cells were gently washed twice with PBS. Calciumchloride （1ml/well） was added and cells were cultured for4h. The calcium-medium wasremoved and cells were washed with pre-cooled PBS and fixed with4%paraformaldehyde（in PBS） for30min，F-actin immunocytochemistry was carried out.Ⅳ Statistics AnalysisStatistics Optical density of F-actin or Calcium immunofluorescence was measureddouble-blindly by using software under magnification of the confocal laser scanningmicroscopy. Results were shown as the mean±S.E. Data analysis was carried out by one-or two-way ANOVA and t-test or Scheffe test or Tamhane test with software SPSS version13.0and a level of p<0.05was considered to be statistical significant.ResultⅠHuman tenocytes have special characteristics phenotypes and can be effectivelystretched on the silicone membranes.1. The isolated cells were characterized as immunopositive for vimentin and collagen Ibut negative for collagen III, indicating these cells were tenocytes.2. It was seen that human tenocytes were grown on the silicone membranes withparallel microgrooves in the surface, which is highly similar to their physiological status.F-actin immunostaining showed no obvious cell shedding even at12%overloading,1.0Hzfor12h, indicating the stretch was effective.ⅡCyclic mechanical stretch can induce calcium overloading1.When the stretch time was set as4h, with the increase of magnitude from0% （unstretch） to4%,8%or12%, significant time-dependant increase of intracellular calciumimmunofluorescence was detected, the highest levels were detected at12%magnitude（p<0.05when compared to other groups）. Similar results were detected when the time wasset at8h. When the time was set as12h, significant increase was detected from0%,4%to8%（p<0.05when compared to0%or4%） then decreased slightly at12%（p>0.05whencompare to8%）. In other words, under baseline （0%magnitude）, the increase of calciumchanged in a time-dependant manner, it significantly increased from4h to8h （p<0.05）, at12h it decreased to the4h level （P>0.05）. Similar results were detected when the magnitudewas set as4%. However, slight differences were detected at8%magnitude, which showedtime-dependant significant increase from4h,8h to12h （p<0.05）. When the magnitude wasset as12%, no significant differences were detected at each timepoint examined （p>0.05）.We further analyzed these data with Two-way ANOVA. The results showed that bothmagnitude and stretch time contributed significantly to the increase of intracellular calciumfluorescent （p<0.01; Tamhane test）.2. Stretch induced intracellular calcium to increase and extracellular calciumoverloading can be inhibited by chelate of extracellular calcium and calcium channel.We used EGTA to block extracellular calcium influx under0.5Hz,8%magnitude.EGTA dramatically decreased intracellular calcium dose-dependently. One-way ANOVAand Tamhane test showed significant differences among untreated,0.5mmol/L and1.0mmol/L EGTA （P<0.05）.MnCl₂was used to put out intracellular calcium fluorescent through to the specificitymechanical sensitivity calcium channel.Tenocytes were stretched by0.5Hz,8%magnitudefor4h. The results showed that0.5mmol and1mmol MnCl₂dramatically inhibitedintracellular calcium in a dose-dependant manner. One-way ANOVA and Tamhane testshowed significant inhibition of calcium by MnCl₂compared to the untreated group（p<0.05）.Under the same stretch conditions as above,20-or40ummol/L nifedipine （L-calciumchannel inhibitor） was applied. The results showed that intracellular calcium increase wasblocked by nifedipine administration in a dose-dependant manner （One-way ANOVA andTamhane test）.3. Extracellular calcium influx and intracellular calcium pool jointly mediate calcium overloading, and calmodulin pathway is not the main factor.Heparin was used to block IP3mediated calcium pool release. The results showed thatheparin induced decrease of intracellular calcium in a dose-dependant manner. One-wayANOVA and Tamhane test showed significant inhibition of calcium by heparinadministration when compared among each treatment （p<0.05）.Phenothiazine is a calmodulin inhibitor. When cells were stretched at0.5Hz,8%magnitude for4h, intracellular calcium concentration was not affected by differentconcentration of phenothiazine （20-,40-or80μmmol/L）（p>0.05; One-way ANOVA,Tamhane test）.4. Repetitive mechanical stretch can activate calcium channel and calcium overloadingmay disrupt cytoskeleton F-actin.To test whether the increased intracellular calcium could be induced by influx ofextracellular calcium, we first set the stretch condition as0.5Hz,8%magnitude and4h,then applied3mmol/L,5mmol/L or10mmol/L CaCl₂to the tenocytes. We found that3mmol/L CaCl₂did not cause significant changes of intracellular calcium compared to thatof the unstretched cells （P>0.05）. However, while intracellular calcium concentration wasslightly upregulated by5mmol/L CaCl₂in unstretched cells, it was dramatically upregulatedby5mmol/L CaCl₂with mechanical overloading. Similar results were noticed by10mmol/LCaCl₂administration, but no significant differences were found between5mmol/L and10mmol/L CaCl₂administration （One-way ANOWA, Tamhane test）.Two-way ANOVA and Tamhane test showed significant differences between3mmol/Land5mmol/L or10mmol/L, no significant differences between5mmol/L and10mmol/Ltreatment （P>0.05）. The above results indicated that cyclic stretch significantly inducesextracellular calcium influx.F-actin immunofluorescence was used to evidence the changes of cytoskeleton undernormal culture conditions （without stretch） with different concentration of CaCl₂treatment.When cells were exposed to3mmol/L CaCl₂for4h, we found that calcium causedcytoskeleton damage in a concentration-dependent manner. Under3mmol/L treatment,slight damages of F-actin of tenocytes were detected and5mmol/L caused cell disshapewith much debris appeared, indicating severe cytoskeleton breakage. Further damages wereseen at10mmol/L, most of the cells became debris. Ⅲ. Mechanical stretch overloading can induce cytoskeleton F-Actin disruptionIn order to examine histological changes of tenocytes under mechanical stretch, weinvestigated the effects of stretch on cell morphology and the expression of F-actin underdifferent stretch strategy. In the unstretched cells, high levels of F-actin immunoreactivitieswere detected, the immunopositive fibers lined in parallel, and the cell morphology wasnormal.When cells were stretched at0.5Hz for4h, their morphology changed, F-actin wasdisorganized under4%magnitude. This injury was further increased by higher stretchmagnitude at8%and12%, F-actin was further decreased, lost or broken, most cells losttheir normal shape.F-actin immunofluorescent staining decreased significantly; filaments wererearranged and became tenuous, thinner, and abnormally distributed. The outline of nucleusbecame unclear and apoptotic cells were observed. One way-ANOVA analysis withTamhane test showed significant decrease of F-actin immunoreactivities at4%or8%magnitude （n=6, p<0.01when compared to that of unstretched cells）; however,12%magnitude did not cause further damage （p>0.05when compare to8%; n=6）We then set the stretch frequency at1.0Hz, and a combination of different magnitudeand time period. The results showed that magnitude but not stretch time contributedsignificantly to the alterations of F-actin immunofluorescent. With the increase ofmagnitude, cell damage was increased and F-actin immunoreactivities were decreasedsignificantly （p<0.01, Two-way ANOVA; Tamhane test）, severe breakage and loss of thesefibers were noticed in most cells.Conclusions1. Mechanical stretch can activate calcium channel opening on tenocytes membrane,induce extracellular calcium influx and trigger release of intracellular calcium, especiallythe inositol triphosphate （IP3）can be caused to induce calcium pool the release is the majorsource of calcium overload; Intracellular calcium overloading may aggravate tendon cellsF-actin injury, and influx of extracellular calcium may be that tendon cells detectmechanical stress which is the signal module of mechanical-biochemical signaltransduction in the early stage.2. Over stretch loading may induce disaggregation，disrupt and rearrangement F-actin of human tendon cells，and its disaggregation and disrupt is positively correlated withstretching force and its duration.3. Cytoskeleton damage, extracellular calcium influx and intracellular calcium poolrelease jointly mediate intracellular calcium overloading, finally lead to calcium depositionin tendon, and, as a result, cause tendon calcification.