Mesenchymal stem cell-conditioned medium accelerates skin wound healing: an in vitro study of fibroblast and keratinocyte scratch assays.

We have used in vitro scratch assays to examine the relative contribution of dermal fibroblasts and keratinocytes in the wound repair process and to test the influence of mesenchymal stem cell (MSC) secreted factors on both skin cell types. Scratch assays were established using single cell and co-cultures of L929 fibroblasts and HaCaT keratinocytes, with wound closure monitored via time-lapse microscopy. Both in serum supplemented and serum free conditions, wound closure was faster in L929 fibroblast than HaCaT keratinocyte scratch assays, and in co-culture the L929 fibroblasts lead the way in closing the scratches. MSC-CM generated under serum free conditions significantly enhanced the wound closure rate of both skin cell types separately and in co-culture, whereas conditioned medium from L929 or HaCaT cultures had no significant effect. This enhancement of wound closure in the presence of MSC-CM was due to accelerated cell migration rather than increased cell proliferation. A number of wound healing mediators were identified in MSC-CM, including TGF-beta1, the chemokines IL-6, IL-8, MCP-1 and RANTES, and collagen type I, fibronectin, SPARC and IGFBP-7. This study suggests that the trophic activity of MSC may play a role in skin wound closure by affecting both dermal fibroblast and keratinocyte migration, along with a contribution to the formation of extracellular matrix.

Walter MN ，Wright KT ，Fuller HR ，MacNeil S ，Johnson WE ... -  《-》

The effect of mesenchymal stem cell conditioned media on corneal stromal fibroblast wound healing activities.

To investigate the effects of conditioned media from mesenchymal stem cells (MSC) on the wound healing activities of corneal stromal fibroblasts. Cell cycle analysis and early stage activation of apoptosis, chemotactic chambers and fibroblast-populated type I collagen gels were used to assess corneal stromal fibroblast proliferation, migration and contraction, respectively. Fibroblasts were obtained from human donor corneas and MSC from fresh rat bone marrow. MSC conditioned media and fibroblast culture medium (FCM), with and without calf serum supplementation, were compared. MSC conditioned media and serum-free FCM had an inhibitory effect on the progression of corneal fibroblasts through the cell cycle. There was a significant increase in the number of cells in the G0-G1 phase for MSC conditioned media and serum-free FCM (p=0.001, p=0.97 respectively). Fibroblast migration and relaxed and stressed gel contraction were significantly inhibited by MSC conditioned media and serum-free FCM compared with FCM with serum (all p=0.001). Glucose and lactate analysis confirmed that these factors were not contributing to this effect. MSC conditioned media was found to inhibit the wound healing activities of corneal stromal fibroblasts in vitro. Putative factors secreted by MSC could be developed for therapeutic use in corneal repair.

Watson SL ，Marcal H ，Sarris M ，Di Girolamo N ，Coroneo MT ，Wakefield D ... -  《-》

Streptolysin O enhances keratinocyte migration and proliferation and promotes skin organ culture wound healing in vitro.

ML-05, a modified form of the hemolytic and cytotoxic bacterial toxin, streptolysin O, is currently being investigated as a treatment for collagen-related disorders such as scleroderma and fibrosis. Furthermore, ML-05 may be effective in promoting wound healing and alleviating the formation of hypertrophic scars and keloids. To investigate the effects of ML-05 on wound-healing processes, in vitro wound-healing scratch assays (using human primary epidermal keratinocytes and dermal fibroblasts) and a human skin organ culture wound model were utilized. ML-05 markedly enhanced keratinocyte migration and proliferation in wound scratch assays. ML-05 did not affect either proliferation or migration of dermal fibroblasts, indicating that ML-05's effects on cell migration/proliferation may be keratinocyte-specific. ML-05 was tested in a dose-dependent manner in a skin organ culture wound model using two different application methods: Through the culture media (dermal exposure) or direct topical treatment of the wound surface. ML-05 was found to accelerate wound healing as measured by reepithelialization, particularly after topical application. Therefore, ML-05 may have potential as a wound-healing agent that promotes reepithelialization through stimulation of keratinocyte migration and proliferation.

Tomic-Canic M ，Mamber SW ，Stojadinovic O ，Lee B ，Radoja N ，McMichael J ... -  《WOUND REPAIR AND REGENERATION》

Proliferation and motility of HaCaT keratinocyte derivatives is enhanced by fibroblast nemosis.

The role of paracrine tumor-stroma regulation in the progression of cancer is under intense investigation. Activated fibroblasts are key components of the tumor microenvironment providing the soluble factors mediating the regulation. Nemosis is an experimental model to study these parameters: formation of a multicellular spheroid activates fibroblasts and leads to increased production of soluble factors involved in the promotion of growth and motility. Role of nemosis was investigated in the tumorigenesis of HaCaT derivatives representing skin carcinoma progression. Conditioned medium from fibroblast spheroids increased proliferation rate of HaCaT derivatives. Expression of proliferation marker Ki-67 increased significantly in benign A5 and low-grade malignant II-4 cells, but did not further increase in the metastatic RT3 cells. Expression of p63, keratinocyte stem cell marker linked to cancer progression, was augmented by medium from nemotic fibroblasts; this increase was also seen in RT3 cells. Scratch-wound healing of the keratinocytes was enhanced in response to fibroblast nemosis. Neutralizing antibodies against growth factors inhibited wound healing to some extent; the response varied between benign and malignant keratinocytes. Migration and invasion were enhanced by conditioned medium from nemotic fibroblasts in benign and low-grade malignant cells. RT3 keratinocyte migration was further augmented, but invasion was not, indicating their intrinsic capacity to invade. Our data demonstrate that fibroblast nemosis increases proliferation and motility of HaCaT keratinocyte derivatives, and thus nemosis can be used as a model to study the role of soluble factors secreted by fibroblasts in tumor progression.

Räsänen K ，Vaheri A 《-》

Extracellular matrix/stromal vascular fraction gel conditioned medium accelerates wound healing in a murine model.

Conditioned medium (CM) is a new treatment modality in regenerative medicine and has shown a successful outcome in wound healing. We recently introduced extracellular matrix/stromal vascular fraction gel (ECM/SVF-gel), an adipose-derived stem cell and adipose native extracellular matrix-enriched product for cytotherapy. This study aimed to evaluate the effect of CM from ECM/SVF-gel (Gel-CM) on wound healing compared with the conventional CM from adipose tissue (Adi-CM) and stem cell (SVF-CM). In vitro wound healing effect of three CMs on keratinocytes and fibroblasts was evaluated in terms of proliferation property, migratory property, and extracellular matrix production. In vivo, two full-thickness wounds were created on the back of each mice. The wounds were randomly divided to receive Gel-CM, Adi-CM, SVF-CM, and PBS injection. Histologic observations and collagen content of wound skin were made. Growth factors concentration in three CMs was further quantified. In vitro, Gel-CM promoted the proliferation and migration of keratinocytes and fibroblasts and enhanced collagen I synthesis in fibroblasts compared to Adi-CM and SVF-CM. In vivo, wound closure was faster, and dermal and epidermal regeneration was improved in the Gel-CM-treated mice compared to that in Adi-CM and SVF-CM-treated mice. Moreover, The growth factors concentration (i.e., vascular endothelial growth factor, basic fibroblast growth factor, hepatocyte growth factor, and transforming growth factor-β) in Gel-CM were significantly higher than those in Adi-CM and SVF-CM. Gel-CM generated under serum free conditions significantly enhanced wound healing effect compared to Adi-CM and SVF-CM by accelerating cell proliferation, migration, and production of ECM. This improved trophic effect may be attributed to the higher growth factors concentration in Gel-CM. Gel-CM shows potential as a novel and promising alternative to skin wound healing treatment. But limitations include the safety and immunogenicity studies of Gel-CM still remain to be clearly clarified and more data on mechanism study are needed.

Deng C ，He Y ，Feng J ，Dong Z ，Yao Y ，Mok H ，Lin M ，Feng L ... -  《-》