Effect Of Mechanical Injury And PGE₂on LOXs And MMP-1,2,3Expression In Co-cultured PCL Fibroblasts And Cynoviocytes

Posterior cruciate ligament （PCL） plays an important role in themaintenance of knee joints stability. However, it is very difficult to fullyrestore the intrinsic biomechanical characteristics after serious injury ofPCL. In the process of PCL injury, mechanical stress not only makes PCLfibroblasts and synoviocytes injured, but also further induces theinflammatory response and the production of inflammatory cytokineswhich could have an effect on PCL fibroblasts and synoviocytes. Cellmigration is one of key steps of injured tissue repair, but the effects of thesecytokines from the inflammatory reaction after PCL injury on the migrationof PCL fibroblasts and synoviocytes have been unclear. It is reported thatmechanical injury and inflammatory cytokines can stimulate both ligamentfibroblasts and synoviocytes to greatly produce matrix metalloproteinases（MMPs）, which may be closely associated with the self-healing ability ofinjured ligament. In addition, lysyl oxidase family （LOXs, including LOXand LOXL1-4） are also involved in the regulation of injured tissue repairprocess through inducing the cross-linking and stabilization of extracellularmatrix. However, so far, the effects of mechanical injury and prostaglandin E₂（PGE₂） derived from the inflammatory reaction after PCL injury onLOXs and MMPs expression in PCL fibroblasts and synoviocytes have notbeen unknown. Furthermore, previous studies on PCL fibroblasts paid littleattention to the influence of the interaction between PCL fibroblasts andsynoviocytes on cell biological characteristics such as cell migration andgenes expression.Objective1. To study the effects of the co-culture environment or PGE₂on themigration of PCL fibroblasts and synoviocytes under mono-culture orco-culture condition;2. To study the effects of12%mechanical injury on LOXs, MMP-1,2,3expressions and MMP-2activity in co-cultured PCL fibroblasts andsynoviocytes;3. To study the effects of PGE₂on LOXs, MMP-1,2,3expressions andMMP-2activity in co-cultured PCL fibroblasts and synoviocytes undermechanical injury.Through above studies, the possible biological mechanism of PCLpoor self-healing ability after injury at the level of cell and molecule wouldbe discussed.MethodsHuman PCL fibroblasts and synoviocytes were mono-cultured orco-cultured using Transwell in vitro. Wound healing assay was performed to determine the migration rate of PCL fibroblasts and synoviocytes inabsence or presence of PGE₂under mono-culture or co-culture. The studyon the effects of mechanical injury and PGE₂on LOXs and MMPsexpression was divided into two parts. The first part mainly studied PCLfibroblasts and were composed of four groups as following. Pâ‘ : themono-culture group for PCL fibroblasts; Pâ‘¡: the co-culture group （PCLfibroblasts were seeded into the lower compartment of Transwell）; Pâ‘¢:co-culture and12%mechanical injury group; and Pâ‘£: the combination ofco-culture,12%mechanical injury and PGE₂. The second part mainlystudied synoviocytes and were also composed of four groups as following.Sâ‘ : the mono-culture group for synoviocytes; Sâ‘¡: the co-culture group（synoviocytes were seeded into the lower compartment of Transwell）; Sâ‘¢:co-culture and12%mechanical injury group; and Sâ‘£: the combination ofco-culture,12%mechanical injury and PGE₂. Total mRNA was isolated at1,3,6and12h after treatments, and quantitative real-time PCR wasemployed to detect LOXs and MMP-1,2,3expressions. The conditionedmedia were collected at12,24,48and72h, and MMP-2activity wasdetermined by zymography.Results1. Compared with the mono-cultured PCL fibroblasts, the co-culturegroup showed better cell status, and the migration rate of co-cultured PCLfibroblasts also showed an approximate2.28-fold increase. But PGE₂ decreased the migration rate of mono-cultured and co-cultured PCLfibroblasts to72%and63%compared with the corresponding control,respectively.2. The co-culture also stimulated synoviocytes to grow and migrate, andthe migration rate of co-cultured synoviocytes showed a1.48-fold increasecompared with the mono-cultured cells. But PGE₂decreased the migrationrate of mono-cultured and co-cultured synociocytes to86%and76%compared with the corresponding control, respectively.3. Compared with the mono-cultured PCL fibroblasts, LOXs expressionshowed a1.24,1.25,1.48,1.66and1.17-fold increase in co-cultured cellsat12h, respectively. But mechanical injury significantly decreased LOXsexpression to64.3%,41.6%,54%,44.6%and19.7%compared with theco-cultured group, respectively. Under co-culture and mechanical injury,PGE₂further decreased LOXs expression to29%,32%,28.4%,27.1%and12%compared with the co-cultured group, respectively.4. Compared with the mono-cultured synoviocytes, LOXs expressionshowed a maximal1.41,1.5,1.66,1.38and1.37-fold increase inco-cultured cells, respectively. But mechanical injury remarkably decreasedLOXs expression to61%,54.5%,51.7%,60.4%and57.7%compared withthe co-cultured group, respectively. Under co-culture and mechanical injury,PGE₂further decreased LOXs expression to52.8%,51.5%,48%,50%and51%compared with the co-cultured group, respectively. 5. Compared with the mono-cultured PCL fibroblasts, MMP-1,2,3expression showed a maximal1.35,1.73and1.45-fold increase inco-cultured cells, respectively. Under co-culture, mechanical injurystimulated MMP-1,2,3expressions to a4,1.67and3-fold increasecompared with the co-cultured group, respectively. Under co-culture andmechanical injury, PGE₂further increased their expression to an8.81,4.2and5.61-fold up-regulation compared with the co-cultured group.6. Compared with the mono-cultured synoviocytes, MMP-1,2,3expression showed a maximal1.78,1.55and1.31-fold increase inco-cultured cells, respectively. Under co-culture, mechanical injurystimulated MMP-1,2,3expression to a1.88,1.17and2.93-fold increasecompared with the co-cultured group. Under co-culture and mechanicalinjury, PGE₂further increased their expression to a2.75,2.01and3.23-foldup-regulation compared with the co-cultured group.7. Compared with the mono-cultured PCL fibroblasts, MMP-2activityshowed a1.92-fold increase at72h in co-cultured cells. Under co-culture,mechanical injury stimulated MMP-2activity to a1.72-fold increasecompared with the co-cultured group. Under co-culture and mechanicalinjury, PGE₂further increased the activity to a6.3-fold up-regulationcompared with the co-cultured group.8. Compared with the mono-cultured synoviocytes, MMP-2activityshowed a1.78-fold increase at72h in co-cultured cells. Under co-culture, mechanical injury stimulated MMP-2activity to a1.5-fold increasecompared with the co-cultured group. Under co-culture and mechanicalinjury, PGE₂further increased the activity to a2.73-fold up-regulationcompared with the co-cultured group.Conclusions1. There is a close cross-talking between PCL fibroblasts andsynoviocytes, and they interact with each other to cooperatively promotecells growth and migration.2. PGE₂significantly inhibited the migration of PCL fibroblasts andsynoviocytes under mono-culture or co-culture.3. The co-culture increased all LOX isoforms expression in both PCLfibroblasts and synoviocytes, and also increased MMP-1,2,3expressionand MMP-2activity.4. Mechanical injury significantly decreased LOXs expression inco-cultured PCL fibroblasts and synoviocytes, but simultaneously increasedMMP-1,2,3expression and MMP-2activity.5. Based on the combined effect of co-culture and mechanical injury,PGE₂further remarkably decreased LOXs expression in both two cells, butsimultaneously further increased MMP-1,2,3expression and MMP-2activity to a greater extent.6. Mechanical injury and PGE₂not only notably inhibited the migration ofPCL fibroblasts and synoviocytes, but also induced the down-regulation of LOXs expression and the up-regulation of MMP-1,2,3expression andactivity. These might be one of possible biological mechanisms of PCLpoor self-healing ability after injury through suppressing cell migration anddisrupting the delicate balance between extracellular matrix biosynthesisand degradation.