Effect Of NPWT On The Development Of Myofibroblast In Diabetic Foot Wound

Background and Objective: Diabetic foot is one of serious complications of diabetes mellitus. Diabetic foot wound, different from non-diabetic wound, often results in delayed wound healing. How to promote the healing of diabetic foot wounds has become an urgent problem to be solved. Negative pressure wound therapy (NPWT) can promote the wound healing. It has been used in the treatment of diabetic foot wounds in recent years.Randomized and controlled trials and systematic reviews have suggested that NPWT is particularly beneficial for the wound healing of diabetic foot compared with conventional treatment. However,the precise mechanisms by which NPWT promotes the wound healing of diabetic foot are still not fully understood. Fibroblasts, one of the major cells in granulation tissue, gradually transform into myofibroblasts during process of wound healing. The main difference between myofibroblasts and fibroblasts is that the myofibroblasts expresses a-smooth muscle actin (a-SMA), which significantly augments contractile activity and is used as an almost universal marker of myofibroblasts. The myofibroblasts are characterized by their capacity to produce force and synthesize more extracellular matrix (ECM) components than fibroblasts, which contribute to remodeling of the granulation tissue. Therefore, myofibroblasts play a more important role during the process of wound healing. Transforming growth factor β1 (TGFβ1) is the major growth factor that facilitates the fibroblast-to-myofibroblast development. The spliced extra domain A fibronectin (EDA+FN) is the most reliable ECM marker of the myofibroblast.This prospective randomized controlled trial evaluated the effect of NPWT on area of the wounds, granulation formation and production of a-SMA, TGFβ1 and EDA+FN in diabetic foot wound and the number of myofibroblast in granulation tissue of diabetic foot wounds,and therefore investigated the effects of NPWT on the development of myofibroblast in diabetic foot wound.Methods:1. From January 2014 to June 2016, 32 patients with diabetic foot wounds fitting the inclusion criteria were enrolled in our clinical trial. The subjects were randomly assigned to negative pressure wound therapy (NPWT) group or control group (Con group) with a computer randomization algorithm. Sixteen patients were treated with NPWT, while the same number of patients in the Con group received advanced moist wound therapy.2. The area of the wounds were measured before and after treatment in both groups,evaluating effect of NPWT and advanced moist wound therapy on wound area in patients with diabetic foot.3. Granulation tissue biopsies were collected before and after treatment from patients with diabetic foot in both groups for hematoxylin-eosin, Masson and immunocytochemical staining, Western blot, Real-time PCR analysis and cell culture.4. The effect of NPWT on myofibroblasts in diabetic foot wound was explored by the following methods in tissue level. The granulation in both groups was evaluated by hematoxylin-eosin and Masson staining. The protein expression of a-SMA, TGFβ1 and EDA+ FN in both groups was assessed by immunocytochemical staining and Western blot analysis. The mRNA expression of a-SMA, TGFβ1 and EDA+ FN in both groups was evaluated by real-time PCR analysis.5. The effect of NPWT on myofibroblasts in diabetic foot wound was studied by the following methods in cell level.The granulation in both groups was collected for cell culture. The percent of myofibroblast of both groups was determined by immunocytochemical staining. The a-SMA, marker of myofibroblasts, was evaluated by Western blot and Real-time PCR analysis.Results:1. There were no significant differences in area of the wounds between after and before treatment in Con group (P>0.05). However, the area of the wounds after treatment in NPWT group was smaller than before treatment (P<0.01).2. (1) The hematoxylin-eosin and Masson staining analysis revealed that although microvascular number and production of collagen in both NPWT and Con group increased after treatment, the difference of microvascular number and production of collagen between after treatment and before treatment in NPWT group was higher than that in Con group (P<0.01). (2) The immunohistochemistry and Western blot analysis showed that although the protein expression of a-SMA, TGFβ1 and EDA+FN increased after treatment in both NPWT and Con group, the difference of the protein expression of a-SMA, TGFβ1 and EDA+FN between after treatment and before treatment in NPWT group was higher than that in Con group (P<0.01). (3) The Real-time PCR analysis showed that although the mRNA expression of a-SMA, TGFβ1 and EDA+FN increased after treatment in both NPWT and Con group, the difference of the mRNA expression of a-SMA, TGFβ1 and EDA+FN between after treatment and before treatment in NPWT group was higher than that in Con group (P<0.01).3. (1) The cell immunohistochemistry analysis showed that the number of myofibroblast enhanced after treatment in both NPWT and Con group. However, the difference of number of myofibroblast between after treatment and before treatment in NPWT group was higher than that in Con group (P<0.01). (2) The western blot analysis revealed that although protein expression of a-SMA increased after treatment in both NPWT and Con group, the difference of protein expression of a-SMA between after treatment and before treatment in NPWT group was higher than that in Con group (P<0.01). (3) The Real-time PCR analysis demonstrated that although mRNA expression of a-SMA increased after treatment in both NPWT and Con group, the difference of mRNA expression of a-SMA between after treatment and before treatment in NPWT group was higher than that in Con group (P<0.01).Conclusion:1. NPWT facilitates granulation formation and reduces area of the wounds than does advanced moist wound therapy.2. NPWT enhances greater protein production of a-SMA, TGFβ1 and EDA+ FN and increases myofibroblast formation in diabetic foot wounds compared to advanced moist wound therapy.3. NPWT promotes production of myofibroblast and formation of granulation, which promotes wound healing of diabetic foot.