Abnormal wound healing encompasses a wide spectrum, from chronic wounds to hypertrophic scars. Both conditions are associated with an abnormal cytokine profile in the wound bed. In this study, we sought to understand the dynamic relationships between myofibroblast differentiation and mechanical performance of the collagen matrix under tissue growth factor-beta (TGF-beta) and tumor necrosis factor-alpha (TNF-alpha) stimulation. We found TGF-beta increased alpha-smooth muscle actin (alpha-SMA) and TNF-alpha alone decreased the basal alpha-SMA expression. When TGF-beta1 and TNF-alpha were both added, the alpha-SMA expression was suppressed below the baseline. Real-time PCR showed that TNF-alpha suppresses TGF-beta1-induced myofibroblast (fibroproliferative) phenotypic genes, for example, alpha-SMA, collagen type 1A, and fibronectin at the mRNA level. TNF-alpha suppresses TGF-beta1-induced gene expression by affecting its mRNA stability. Our results further showed that TNF-alpha inhibits TGF-beta1-induced Smad-3 phosphorylation via Jun N-terminal kinase signaling. Mechanical testing showed that TNF-alpha decreases the stiffness and contraction of the lattices after 5 days in culture. We proposed that changes in alpha-SMA, collagen, and fibronectin expression result in decreased contraction and stiffness of collagen matrices. Therefore, the balance of cytokines in a wound defines the mechanical properties of the extracellular matrix and optimal wound healing.

Background Metalloproteinase-9 (MMP-9) is a type IV Collagenase found at elevated levels in chronic wounds. As wounds heal, MMP-9 diminishes. In this study, we investigated whether MMP-9 directly contributes to chronic wound pathogenesis. Methods Recombinant proMMP-9 was prepared using immortalized keratinocytes transduced by a lentivirus. ProMMP-9 was purified from cell culture media and activated using 4-aminophenylmercuric acetate. Active MMP-9 was then suspended in Xanthan Gum to a concentration paralleling that found in human chronic wounds. Two parallel 6mm punch biopsies were made on the backs of C57BL mice. Wounds were treated daily with MMP-9 or vehicle. Wound areas were measured and tissues examined by densitometry, real-time RT-PCR, histology, and immunohistochemistry at days 7, 10 and 12. Results Exogenous MMP-9, at the level found within chronic wounds, delayed wound healing in this animal model. By 7 days, wounds in the MMP-9-injected group were 12% larger than control wounds (p=0.008). By day 12, wounds in the MMP-9-injected group were 25% larger than those of the control group (p=0.03). Histologic examination shows that high levels of active MMP-9 impaired epithelial migrating tongues (p=0.0008). Moreover, consistent with elevated MMP-9, the collagen IV in the leading edge of the epithelial tongue was diminished. Conclusion MMP-9 appears to directly delay wound healing. Our data suggests that this may occur through interference with re-epithelialization. We propose that MMP-9 interferes with the basement membrane protein structure, which in turn impedes keratinocyte migration, attachment, and the reestablishment of the epidermis.

Background:  The cytokine signals that regulate myofibroblast differentiation and the resulting reorganization of matrix are incompletely understood. We studied the response of fibroblasts to mediators known to affect matrix reorganization, TGF‐β and TNF‐α in a collagen matrix. Further, we sought to determine if any cell phenotype changes resulted in quantifiable alterations in the mechanical performance of the matrix. Methods:  Normal human dermal fibroblasts were cultured in collagen type I lattices for 1 and 5 days in DMEM with .5% FBS and TGF‐β(1 ng/ml), TNF‐α(10 ng/ml), or combinations TGF‐β(1 ng/ml) + TNF‐α(1,5, and 10 ng/ml). Western blot and immunohistochemistry were used to determine α‐SMA expression. Quantitative real‐time PCR was used to measure α‐SMA mRNA. An indentation mechanical method was used to measure Young’s modulus which represents matrix stiffness. Results:  TGF‐β increases α‐SMA, while TNF‐α alone has no effect on basal α‐SMA expression. When TGF‐β + TNF‐α were both added, the expression of α‐SMA returned to basal levels. Immunohistochemistry showed α‐SMA is incorporated into prominent stress fibers only in the TGF‐β group. Real‐time PCR showed that TNF‐α did not affect α‐SMA mRNA, while TGF‐β increased α‐SMA mRNA 19‐fold. Combination treatment reduced mRNA level by 16‐fold, to 2.7‐fold compared to control. Indentation testing showed that, in all treatment groups, lattices after 5 days in culture were stiffer. TNF‐α appeared to reduce the stiffness of the lattices on both day 1 and 5 compared to control. Conclusion:  TNF‐α decreases TGF‐β induction of α‐SMA expression at the transcriptional level and decreases lattice stiffness. The cellular responses to cytokines are reflected in both biochemical and mechanical behaviors. Understanding both will be necessary to understand how tissues remodel into functional tissue and scar.