Experimental Research Of Directional Contraction Of Fibroblast Populated Collagen Lattice Induced By Direct Current Electric Fields

Background and purpose:Contracture deformity of hypertrophic scaring is a commom complication after wound healing of deep dermal burn injury,which not only affects the appearance,but also leads to the movement disorder of limb joints and functional parts,and even a permanent loss of function.It is a key to restrict the improvement of burn patients' life quality and has become a major scientific problem of burn which has not been solved.Previous studies showed that the continued contraction of scar tissue results in contracture deformity of hypertrophic scaring which on the one hand attributes to proliferating fibroblasts and collagen fibers of excessive sedimentation,and on the other hand relates to the array structure of fibroblasts and collagen fiber in scar tissue.Scanning electron microscope observation found that fibroblasts and its extracellular collagen fiber are parallel to the tention lines of the scar,which can be speculated that cell coupls the extracellular collagen fibers by matrix molecules,and continued to produce and maintain directional contractive tension,is a key pathological structure of scar contracture.Previous studies have important implications for revealing the cellular and molecular mechanisms of scar contracture.However,as lack of effective model that simulate the dynamic process of scar contracture of clinical patients in vitro,the understanding of scar contracture is confined to tissue slice observation and theoretical analysis,the cellular and molecular mechanisms of scar contracture were not well elucidated.In 1979,Bell established the fibroblast populated collagen lattice(FPCL)by culturing and mixing rat tail collagen and fibroblasts together.For the first time FPCL was used as a model to simulate scar tissue contraction in vitro,the model reveals that the fibroblasts and myofibroblasts are the main effectors of scar tissue contraction and found that the concentrations of fibroblast and collagen effectd the rate and final degree of FPCL contraction.Using this model,Ehrlich further found that the activation of rapid myosin ATPase remodeling microfilaments and causing cytoplasmic microfilaments combination with collagen fibrils,directing the arrangement of collagen fibrils and starting wound contraction.With the development of wound contractive tension,the continuous tense of stress fiber maintaining wound contractive tension by the activation of sustained myosin ATPase,the above study shows that cytoskeleton-actin stress fibers plays an important role in the maintenance of continued contraction of FPCL,and deeply understand more characteristics of the scar tissue contraction.However,nearly 30 years,the FPCL model did not help people to make a breakthrough in the study and control of scar contracture deformities,the fundamental reason is that the model does not simulate the most important feature of scar tissue directional contraction of clinical cases.The established model of Bell is concentric contraction,fibroblasts are distributed randomly arranged,which is essentially different from directional contraction and pathologic structural of scar tissue of joints in vivo,that fibroblasts coupling collagen fibers in parallel with the direction of the scar contracture and thus FPCL does not simulate the ideal model of directional contraction of scar tissue.Bioelectric field is widespread from lower mircrobes to higher mammals,which is a common phenomenon in the biosphere.Reserch shows that bioelectric field plays an important role in the prosess of wound healing,tissue regeneration,and embryonic ontogeny.Endogenous direct current electric field are generated instantaneously after an injury due to the disappaer of the trans-epithelial potentials,with the wound center being more negative than the surrounding skin tissue and thus acting as the cathode of the endogenous EFs until the wounds healed eventually,and the electric field intensity are between 50 and 200 m V/mm.It is of significance to cell morphology,movement,proliferation,migration and orientation of wound healing,and is important biological signals of wound healing.Studies have found that the physiologic strength of exogenous DC electric field can induce dermal fibroblasts of human,mice and rat vertically aligning the vector direction of electric field,it is help to repair normal morphological structure of damage organization.However,different kinds of fibroblasts present various biological reactivity response to exogenous DC electric field.Within EF hour 24,threshold voltage of perpendicular to electric field vector of human dermal fibroblast is 100mV/mm,while increase to 200 m V/mm in NIH 3T3 mouse in hour 3.The roles of physiologic strength of exogenous DC electric fields inducing fibroblasts alignment are time and voltage dependence.This study was based on a characteristic of fibroblasts orientation in the DC electric field,to explore directional contraction of FPCL induced by electric fields,to develop an ideal model of directional contraction of scar tissue,it is a good theoretical and experimental basis for further research of the cellular and molecular mechanisms of scar contracture,and developing a new prevention and treatment strategies of scar contracture.Contents and methods:1.Research of exogenous DC electric field induce fibroblasts alignment in 2D cultivationEstablish an application device of exogenous DC electric field,which is applied to observe the effects of fibroblasts alignment by different time and voltage of DC electric field.2.Research of exogenous DC electric field induce fibroblasts alignment in 3D cultivationEstablish an 3D application device of exogenous DC electric field,which is applied to observe the effects of fibroblasts alignment by different time and the electric field strength of DC electric fields.3.Experimental Research of Directional Contraction of Fibroblast Populated Collagen Lattice Induced by Direct Current Electric FieldsAt first,we should confirm the fibroblast density which one is appropriate of inducing FPCL contraction,then we can observe effects of directional contraction of FPCL induced by exogenous DC electric fields.4.Explore the skeleton mechanisms of directional contraction of FPCL induced by exogenous DC electric fields.Confirm the effects of skeleton structure of microfilaments and microtubulin in directional contraction of FPCL and fibroblast alignment induced by exogenous DC electric field by depolymerizing or promoting skeleton.Results:1.Fibroblasts are random distribution without exogenous DC electric field in 2D cultivation,and the orientation index is-0.02 ? 0.10,while fibroblasts are vertically aligning the vector direction of electric field in 100mV/mm for 4 h,and the orientation index is 0.56? 0.07.The roles of DC electric field inducing fibroblasts alignment are time and voltage dependence.2.The roles of DC electric field inducing fibroblasts alignment of 3D cultivation are time and voltage dependence,and the best effects of inducing environment is 300mV/mm for 4 h,the orientation index is 0.69 ? 0.06.3.Establish an model of contraction of FPCL successfully.The fibroblasts of FPCL are random distribution,and the centripetal contraction of FPCL is cell density dependence.4.Exogenous DC electric field(300mV/mm for 4 h)could induce fibroblasts aligning the vector direction of electric field,and then induce directional contraction of FPCL successfully,that is enhancing contraction of the vector direction of electric field,and the sametime wenkening contraction of the parallel direction of electric field.5.Exogenous DC electric field has no significant effect on the morphology and distribution of microfilaments and microtubulins.Depolymerizing microfilaments by cytochalasin D is significance of inhibiting directional contraction of FPCL induced by exogenous DC electric field,while depolymerizing microtubulins by colchicine has no significant effect on it.6.Depolymerization of microfilaments and microtubulins have no significant effect on fibroblasts alignment induced by exogenous DC electric field.Enhancing or inhibition of stress fiber by phosphorylation,non phosphorylated HSP27 with a denovirus vector has no significant effect on fibroblasts alignment induced by exogenous DC electric field.Conclusion:1.Exogenous DC electric field could induce fibroblasts alignment in 2D or 3D cultivation,and cause directional contraction of FPCL.induced by exogenous DC electric field,and be used to developing a new prevention and treatment strategies of an vitro model of directional contraction of clinical scar tissue.2.Fibroblasts alignment is an important basis of cell morphology for directional contraction of FPCL,the skeleton structure of microfilament is a key to regulate directional contraction of FPCL.3.Depolymerization or polymerization of microfilaments and microtubulins have no significant effect on fibroblasts alignment induced by exogenous DC electric field.4.Directional contraction of FPCL induced by exogenous DC electric fields,providing a model of prevention and treatmenting of directional contraction of scar tissue in vitro.5.Alignment of fibroblasts could be regulated by exogenous DC electric fields,and then could be used to developing a new prevention and potential treatment strategies of directional contraction of scar tissue of joints in vivo.