Study On The Mechanobiologic Effects Of The Pressure On BMSCs With A Newly Developed Hydrolic Cellular Pressure Unit

Temporomandibular joint (TMJ) is one of the most intricate and complicated loadingjoints in the human body, and the motivation of its growth and remodeling results from thebearing. Condylar cartilage is the stress-sensitive load bearing area, which plays a key rolein joint movement. Though the thickness is limited, condylar cartilage not only has highcompressive strength and elasticity, but also disperses stress during movement. Thedefects, degeneration, even necrosis of condylar cartilage are very common in clinics, andmost of them are caused by inflammation, tumor, trauma and dysplasia. However, therepair and regeneration of condylar cartilage is extremely difficult. For one thing, slowmetabolism and lacking of blood supply, the condylar cartilage needs constant mechanicalstimulation to maintain the integrity of its structure and function. For the other, this kind ofpressure exists throughout the joint movement and ranges from compressive pressure tonegative with the change of mode and range of motion. Therefore, for condylar cartilage，the extremely complicated mechanical environment is crucial for its remodeling andregeneration. Among the stem cells having chondrogenic potential, bone marrow mesenchymalstem cells (BMSCs) is superior to periosteum derived stem cells for easily obtainable, toadipose-derived stem cells (ADSCs) for higher osteogenic potential and to embryonicstem cell (ESC) for involving less ethical concerns.Thus, the most effective method torepair the cartilage defect was either transplanting the combination of BMSCs and scaffoldmaterials into defect areas or inducing the endogenous BMSCs homing to the injured site.However, the main limitation of this method is that the mechanical strength of newlyformed extracellular matrix (ECM) is insufficient. Previous research has shown that themechanical stimulation itself could induce BMSCs to differentiate into chondrocytes andfurther formed cartilage tissues rich in matrix. Some scholars proposed that mechanicalstimulation is essential for chondrogenic differentiation of BMSCs for it could inhibit thehyperplasia and hypertrophy of chondrocytes to make sure the mechanical features ofnewly formed cartilage tissues. Therefore, imitating the growth-microenviroment ofBMSCs in vitro and controlling that in vivo are both necessary for cartilago articularisregeneration.But in fact, in the process of mandibular movement, the intra-articularpressureendures a relative large range of variation, which makes the outcomes followingremodeling and regeneration hard to handle. By now, no mechanical device could mimicsuch complicated mechanical environment, so that the biomechanical regulation effect inthe process of repair and regeneration of condylar cartilage in such a peculiar mechanicalenvironment and its mechanism are still unknown.In view of the above mentioned problems, the present study, based on the controlledhydraulic loading device we have made, develops a new device which could convert thepressure form compressive to negative and control temperature. This new device couldrealize imitation of different mode of pressures on BMSCs, including static or dynamic,and compressive or negative, and then the proliferation, cell cycle, ultrastructure,cytoskeleton, apoptosis, chondrogenic differentation of BMSCs were conducted toevaluated the biomechanical effect of mechanical environment of TMJ on stem cells. Theresearch in this paper will provide basic theories and criterions in applications for cartilagoarticularis tissue engineering. The content of this item includes two parts summarized asfollowing. 1. Development of a new multi-funtional hydrolic cellular pressure unitThe system consisted of three parts: cell culture system, loading control system anddata processing system. Different mode of pressures could be loaded on the cells and aseries of biological effects of stem cells were further evaluated. The parameter setting wasshown as follows: the pressure ranged from-50to300KPa, the accuracy of dynamicpressure was controlled within±5%，the accuracy of static pressure was controlled within±1%for negative while±3%for compressive, the temperature should be within36±2℃,and the frequency of load ranged from0.01Hz to0.1Hz.This device could overcome the temperature compensation causing by different kindsand different ranges of pressure by using the combination of thermostatic water bath andauxiliary heating device to maintain the temperature for cell culture. It could provide arelative large range of pressure using a combined loading system, and could monitor everychanges of pressure and temperature inside of incubator in real time by using monitoringsoftware. Besides, this system also has some other characteristics, such as easy-to-handle,highly control precision and stability, multiple pressure modes, reliable performance andso on, which is suitable for the research of other types of stress-sensitive cells (i.e.articular chondrocytes, osteoblast, periodontal ligament cells and so on) as well.2．Mechanobiological effects of BMSCs to different types of pressures.In experiment1, the BMSCs in rats were isolated and cultured via the method ofwhole bone marrow adherent. They were proved to be the mesenchyme stem cells bymeans of drawing the cell growth curve as well as identifying the surface markers and theability of multi-differentiation.In experiment2, the BMSCs were treated with5types of pressure produced by thenovel developed cytomechanics loading device in vitro, including static negative pressure（10KPa、-20KPa、-30KPa、-40KPa、-50KPa）、dynamic negative pressure（0~10KPa、0~20KPa、0~30KPa、0~40KPa、0~50KPa，0.1Hz）、static compressivepressure（45KPa、90KPa、135KPa、180KPa）、dynamic compressive pressure（0~45KPa、0~90KPa、0~135KPa、0~180KPa，0.1Hz） and dynamic compressive-negativepressure（-20~45KPa、-20~90KPa、-20~135KPa、-20~180KPa、-40~45KPa、-40~90KPa、-40~135KPa、-40~180KPa，0.1Hz）. The above26groups of BMSCs were treated with pressure1h/d for consecutive2days, while the control BMSCs were treatedwithout pressure. CCK8was used for detecting the proliferation and flow cytometry(FCM) was used for detecting the cell circle. The results showed us that, certain pressurecould improve the cell division and proliferation of BMSCs. The dynamic pressure has abetter effect on promoting the proliferation than the static pressure. Among the dynamicpressures, the low negative pressure and high compressive pressure were preferred, whilethe pressure of-40~135KPa was the best to cell proliferation in the dynamiccompressive-negative pressure. Based on the results, the following12sub-types ofpressures（static pressure,-20KPa、-40KPa; dynamic negative pressure,0~20KPa、0~40KPa，0.1Hz; static compressive pressure,45KPa、90KPa; dynamic compressivepressure,0~45KPa、0~90KPa，0.1Hz; dynamic compressive-negative pressure,-20~45KPa、-20~90KPa、-40~45KPa、-40~90KPa，0.1Hz） were selected to be tested inthe following study.In experiment3, transmission electron microscopy (SEM) and laser confocalmicroscope were used to observe the ultrastructure and cytoskeleton of BMSCs after beingtreated with different types of pressures. The character of exuberant protein secretion, likeendoplasmic reticulum expansion and abundant microvilli, occurred in the BMSCs treatedwith low static negative pressure (-20KPa) or dynamic negative pressure, while theBMSCs treated with high negative pressure (-40KPa) or dynamic negative pressureshowed early apoptosis. The BMSCs treated with low static compressive pressure (45KPa)or dynamic compressive pressure showed mild expansion of endoplasmic reticulum, neatarrangement of mitochondrial cristae, abundant ribosome and microvilli, and secretedcollagen around the cell. However, the BMSCs treated with high static pressure ordynamic pressure showed the character of apoptosis coinciding with rich matrix,indicating that this sub-type pressure could promote renewing the BMSCs and result inmore cell apoptosis, with the concurrence of acceleration of the cell differentiation processand the increase of secretion capacity. The large nucleolus, expansive endoplasmicreticulum and good cell viability were only observed in the BMSCs in the-40~90KPagroup, while the BMSCs in the other dynamic compressive-negative groups showeddifferent levels of cell apoptosis. The results of F-actin fluorescence staining showed us that, static negative pressure, dynamic negative pressure, static compressive pressure anddynamic compressive pressure could promote F-actin expression and stress fiber assemblyeffectively. Also, the stress fiber assembly could be the results of dynamiccompressive-negative pressure stimuli, while the staining degree F-actin was lower underthis kind of stimuli compared with the stimuli of dynamic compressive pressure anddynamic negative pressure.In experiment4, the cell apoptosis was tested via FCM. The amount cell apoptosis ofBMSCs in the static negative pressure（-40KPa）group and dynamic compressive-negativepressure group（-20~90KPa、-40~45KPa）significantly increased, which may attributeto the reason that the continuous high-strength hydrostatic pressure has overcome thephysiological loading of BMSCs. In this study, the apoptosis of BMSCs increased afterlow dynamic negative pressure（0~-20KPa）, high static compressive pressure（90KPa）,high dynamic compressive pressure （0~90KPa） and certain range of dynamiccompressive-negative pressure （-40~90KPa）stimuli which were proved to have thepotential to promote cell proliferation by experiment2. These findings suggest that cellapoptosis may be one of patterns that self–remodeling and self-adjustment, and it isconsistent with the changes observed in the cell microstructure. The cyclic hydrostaticpressure with small stress variation scope might not lead into the change of biologicalcharacter like cell proliferation and apoptosis because of insufficient effects.In experiment5, chondroblast marker genes of BMSCs treated with12sub-types ofpressure from static negative pressure, dynamic negative pressure, static compressivepressure, dynamic compressive pressure and dynamic compressive-negative pressure weretested via Real-time PCR. We found that low dynamic negative pressure（-20KPa）andstatic negative pressure（0~-20KPa）promoted the Sox9and Aggrecan expression inBMSCs significantly, while high dynamic negative pressure（-40KPa）and static negativepressure（0~-40KPa）inhibited the expression of chondroblast marker genes. High staticcompressive pressure（90KPa）and dynamic compressive pressure（0~90KPa） promotedthe expression of all the chondroblast marker genes, including Sox9, Aggrecan andcollagen type1I, while low compressive pressure（45KPa）and dynamic compressivepressure（0~45KPa）only promote the Sox9expression. Only the BMSCs treated with dynamic compressive-negative pressure （-40~90KPa）could increase the expression ofall the chondroblast marker genes, including Sox9, Aggrecan and collagen type1I, but notfor the other three sub-types of dynamic compressive-negative pressure although the Sox9and Aggrecan expressions could be promoted to some extent. Among all the five types ofpressures, dynamic pressure has a better capability of promoting cartilage formation thanstatic pressure, with the fact that0~90KPa is the most suitable stimuli followed by-40~90KPa.SummaryThe present study developed a new type of multi-functional device which was used toload different types of pressure on cells in vitro. This was the first time for one unit toimitate diversified kinds of pressures at the same time. This new device has manycharacteristics, such as easy-to-handle, highly control precision and stability, multiplepressure modes, reliable performance and so on. Then, the proliferation, cell cycle,ultrastructure, cytoskeleton, apoptosis and the chondrogenic differentiation potential ofBMSCs were evaluated after being loaded different pressures conditions to reveal thebiomechanics of pressure on BMSCs. The results demonstrated that the biologicalproperties of BMSCs could be activated by the dynamic and negative pressure with lowintensity （0~-20KPa）, static or dynamic compressive pressure with high intensity（90KPa、0~90KPa）, and dynamic pressure ranging from-40to90KPa. However, thedynamic and negative pressure ranging from0to-20KPa could not up-regulate theexpression of Col-II, while the apoptosis was also obvious in static compressive pressurewith the intensity of90KPa.Hence, only dynamic pressures ranging from0to90KPa andranging from-40to90KPa play an overall promoting effect on cells’ biological functions,and the latter, particularly promoted chondrogenic of BMSCs.Based on the complexity of the mechanical environment for condylar cartilage, thisstudy revealed the biomechanical effect of different types of pressures on the biologicalfunctions. The present study will provide basic theories and criterions in applications forcartilago articularis tissue engineering, and shed some light on future researches aboutother pressure-sensitive cells in vitro.