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Optimum healing of a cutaneous wound requires a well-orchestrated integration of the complex biological and molecular events of cell migration and proliferation, and of extracellular matrix deposition and remodeling. Several studies in recent years suggest that bone marrow derived stem cells such as mesenchymal stem cells, progenitor cells such as endothelial progenitor cells and fibrocytes may be involved in these processes, contributing to skin cells or releasing regulatory cytokines. Stem/progenitor cells may be mobilized to leave the bone marrow, home to injured tissues and participate in the repair and regeneration. Direct injection of bone marrow derived mesenchymal stem cells or endothelial progenitor cells into injured tissues shows improved repair through mechanisms of differentiation and/or release of paracrine factors. Enhanced understanding of these cells may help develop novel therapies for difficult cutaneous conditions such as non-healing chronic wounds and hypertrophic scarring as well as engineering cutaneous substitutes.

Study Type – Aetiology (case control)
Level of Evidence 3b OBJECTIVE To investigate the progression of urodynamic changes, as well as histological and biochemical outcomes over a prolonged period of partial bladder outlet obstruction (pBOO) in an animal model with physiologically relevant pBOO. MATERIALS AND METHODS Healthy, adult, female Fischer rats underwent surgical creation of a pBOO for either 2, 4, 8, or 13 weeks and were compared with sham‐operated rats. Urodynamic measurements were used to compare bladder volumes and pressure. Tissue was grossly analysed with light microscopy and bladder weights and thicknesses were compared. Reverse transcription‐polymerase chain reaction for collagen, transforming growth factor β (TGF‐β), connective tissue growth factor (CTGF), hypoxia inducible factor 1α (HIF‐1α), and platelet‐derived growth factor (PDGF‐A) was performed on all samples, as well as immunohistochemistry (IHC) for α‐smooth muscle actin (α‐SMA). Finally, mass spectrometry was used to quantify the collagen content of the bladders as a measure of fibrosis. RESULTS After induction of pBOO, all rats remained healthy. Initial urodynamics showed an increase in capacity while maintaining normal pressures, but then deteriorated into small capacity, high‐pressure bladders. Haematoxylin and eosin (H&E) staining showed an initial inflammatory response, and this was confirmed with significantly increased mRNA levels of TGF‐β, CTGF, HIF‐1α, and PDGF. The progression to smooth muscle hypertrophy was evident on H&E and confirmed with increased bladder mass and thickness. IHC for α‐SMA showed a progressive increase associated with the elevated bladder pressures. Masson’s trichrome and mass spectrometry showed a progressive increase in collagen to 13 weeks. CONCLUSION With this model, we have effectively replicated the clinical scenario, with significant pathophysiological changes occurring insidiously in otherwise healthy rats. We believe that our observed changes represent distinct phases of bladder decompensation; with an initial inflammatory response to the stress of the pBOO, smooth muscle hypertrophy to overcome the increased urethral resistance, and eventual decompensation to fibrosis. The time course of the inflammatory markers implies the need for early intervention to prevent this cascade. Novel strategies targeting these observed physiological responses could lead to improved preventative strategies, with respect to biochemical pathways and the time course of their initiation.

Hypertrophic scar (HTS) following thermal injury is a dermal fibroproliferative disorder that leads to considerable morbidity. The development of HTS involves numerous cell types and cytokines with dermal fibroblasts being a key cell. We have previously reported that the phenotype of fibroblasts isolated from HTS was altered compared to fibroblasts from normal skin. In this study, normal skin was horizontally sectioned into five layers using a dermatome from which fibroblasts were isolated and cultured. Cells from the deeper layers were observed to proliferate at a slow rate, but were morphologically larger. In ELISA and FACS assays, cells from the deeper layers produced more TGF-b1 and TGF-b1 producing cells were higher. In quantitative RT-PCR, the cells from the deeper layers had higher CTGF and HSP47 mRNA levels compared to those from superficial layers. In western blot, FACS and collagen gel assays, fibroblasts from the deeper layers produced more a-smooth muscle actin (a-SMA), had higher a-SMA positive cells and contracted collagen gels more. Fibroblasts from the deeper layers were also found to produce more collagen, but less collagenase by mass spectrometry and collagenase assay. Interestingly, cells from the deeper layers also produced more of the proteoglycan, versican, but less decorin. Taken together, these data strongly demonstrate that fibroblasts from the deeper layers of the dermis resemble HTS fibroblasts, suggesting that the deeper layer fibroblasts may be critical in the formation of HTS. Hypertrophic scarring is a common fibroproliferative disorder of the human dermis characterized by erythematous, raised, pruritic lesions of the healing skin, which is usually following thermal and other injuries that involve the deep dermis. 1 These lesions lead to scarring that compromises the appearance of healing skin and are commonly associated with contractures that limit movement and function of involved joints and facial features. As such, HTS is the principal factor that contributes to the prolonged and often uncomfortable rehabilitation period for thermally injured patients, particularly those who have survived large lifethreatening injuries, children, and individuals of dark-skinned races, in whom such scarring occurs more commonly. 2 The undesirable physical properties of HTS tissue can be attributed to the presence of a large amount of extracellular matrix that is of altered composition and organization, compared to normal dermis or mature scar. This matrix is the product of a dense population of fibroblasts, maintained in a hyperactive state by inflammatory cytokines such as TGF-b and other factors, some of which may be physical in origin. 3 Although the molecular and cellular events that lead to HTS have been extensively studied, the pathogenesis of this condition is still not well understood, making treatment difficult.Key cells involved in HTS are the dermal fibroblasts. Dermal fibroblasts are a dynamic and diverse population of cells whose function in skin and HTS in many respects remain unkn...

Hypertrophic scar (HTS), a fibroproliferative disorder (FPD), complicates burn wound healing. Although the pathogenesis is not understood, prolonged inflammation is a known contributing factor. Emerging evidence suggests that fibroblasts regulate immune/inflammatory responses through toll-like receptor 4 (TLR4) activated by lipopolysaccharide (LPS) through adaptor molecules, leading to nuclear factor kappa-light-chain-enhancer of activated B cells and mitogen-activated protein kinases activation, cytokine gene transcription and co-stimulatory molecule expression resulting in inflammation. This study explored the possible role of TLR4 in HTS formation. Paired normal and HTS tissue from burn patients was collected and dermal fibroblasts isolated and cultured. Immunohistochemical analysis of tissues demonstrated increased TLR4 staining in HTS tissue. Quantitative RT-PCR of three pairs of fibroblasts demonstrated mRNA levels for TLR4 and its legend myeloid differentiation factor 88 (MyD88) in HTS fibroblasts were increased significantly compared with normal fibroblasts. Flow cytometry showed increased TLR4 expression in HTS fibroblasts compared with normal. ELISA demonstrated protein levels for prostaglandin E2, interleukin (IL)-6, IL-8 and monocyte chemotactic protein-1 (MCP-1) were significantly increased in HTS fibroblasts compared to normal. When paired normal and HTS fibroblasts were stimulated with LPS, significant increases in mRNA and protein levels for MyD88, IL-6, IL-8, and MCP-1 were detected. However, when transfected with MyD88 small interfering RNA (siRNA), then stimulated with LPS, a significant decrease in mRNA and protein levels for these molecules compared to only LPS-stimulated fibroblasts was detected. In comparison, a scramble siRNA transfection did not affect mRNA or protein levels for these molecules. Results demonstrate LPS stimulates proinflammatory cytokine expression in dermal fibroblasts and MyD88 siRNA eliminates the expression. Therefore, controlling inflammation and manipulating TLR signaling in skin cells may result in novel treatment strategies for HTS and other FPD.