Mesenchymal stem cell‐conditioned medium accelerates wound healing with fewer scars

Mesenchymal stem cells (MSCs) derived from umbilical cord s (UC‐MSCs) have been shown to enhance cutaneous wound healing by means of the paracrine activity. Fibroblasts are the primary cells involved in wound repair. The paracrine effects of UC‐MSCs on dermal fibroblasts have not been fully explored in vitro or in vivo. Dermal fibroblasts were treated with conditioned media from UC‐MSCs (UC‐MSC‐CM). In this model, UC‐MSC‐CM increased the proliferation and migration of dermal fibroblasts. Moreover, adult dermal fibroblasts transitioned into a phenotype with a low myofibroblast formation capacity, a decreased ratio of transforming growth factor‐β1,3 (TGF‐β1/3) and an increased ratio of matrix metalloproteinase/tissue inhibitor of metalloproteinases (MMP/TIMP). Additionally, UC‐MSC‐CM‐treated wounds showed accelerated healing with fewer scars compared with control groups. These observations suggest that UC‐MSC‐CM may be a feasible strategy to promote cutaneous repair and a potential means to realise scarless healing.     

Transdifferentiated circulating monocytes release exosomes containing 14‐3‐3 proteins with matrix metalloproteinase‐1 stimulating effect for dermal fibroblasts

Fibroblasts are major cellular components of healing wounds. In this regard, it remains to be fully understood how different paracrine signals may influence the final collagen/matrix metalloproteinase (MMP) balance in resident fibroblasts. Our previous reports have demonstrated that circulating stem cells and monocytes can be transdifferentiated into “keratinocyte‐like cells” under certain culture conditions. These transformed cells are able to stimulate MMP‐1 expression in dermal fibroblasts. However, the underlying mechanism of this cell‐to‐cell interaction is unknown. This study describes exosomes as a major delivery system that keratinocyte‐like cells use to release proteins into the conditioned media. The exosomes exhibited distinctive size, density, and saucer‐like morphology. Using PKH‐26 and GFP‐adenovirus infection, we demonstrated that exosomes are able to fuse and then release their protein content into dermal fibroblasts. Mass spectrometry and Western blotting identified five 14‐3‐3 isoforms (β, γ, ?, τ, and ζ) as MMP‐1 stimulating factors for dermal fibroblasts. Immunoprecipation assays confirmed that these 14‐3‐3 isoforms account for almost the entire MMP‐1 up‐regulation induced by exosomes. In summary, our results demonstrated that circulating monocytes stimulated to be transformed into “keratinocyte‐like cells” could promote an anti‐fibrogenic commitment of dermal fibroblasts via exosomal 14‐3‐3 proteins.     

Cell surface engineering using glycosylphosphatidylinositol anchored tissue inhibitor of matrix metalloproteinase‐1 stimulates cutaneous wound healing

The balance between matrix metalloproteinases and their endogenous tissue inhibitors (TIMPs) is an important component in effective wound healing. The biologic action of these proteins is linked in part to the stoichiometry of TIMP/matrix metalloproteinases/surface protein interactions. We recently described the effect of a glycosylphosphatidylinositol (GPI) anchored version of TIMP‐1 on dermal fibroblast biology. Here, cell proliferation assays, in vitro wound healing, electrical wound, and impedance measurements were used to characterize effects of TIMP‐1‐GPI treatment on primary human epidermal keratinocytes. TIMP‐1‐GPI stimulated keratinocyte proliferation, as well as mobilization and migration. In parallel, it suppressed the migration and matrix secretion of dermal myofibroblasts, and reduced their secretion of active TGF‐β1. Topical application of TIMP‐1‐GPI in an in vivo excisional wound model increased the rate of wound healing. The agent positively influenced different aspects of wound healing depending on the cell type studied. TIMP‐1‐GPI counters potential negative effects of overactive myofibroblasts and enhances the mobilization and proliferation of keratinocytes essential for effective wound healing. The application of TIMP‐1‐GPI represents a novel and practical clinical solution for facilitating healing of difficult wounds.     

Acceleration of cutaneous wound healing by brassinosteroids

Brassinosteroids are plant growth hormones involved in cell growth, division, and differentiation. Their effects in animals are largely unknown, although recent studies showed that the anabolic properties of brassinosteroids are possibly mediated through the phosphoinositide 3‐kinase/protein kinase B signaling pathway. Here, we examined biological activity of homobrassinolide (HB) and its synthetic analogues in in vitro proliferation and migration assays in murine fibroblast and primary keratinocyte cell culture. HB stimulated fibroblast proliferation and migration and weakly induced keratinocyte proliferation in vitro. The effects of topical HB administration on progression of wound closure were further tested in the mouse model of cutaneous wound healing. C57BL/6J mice were given a full‐thickness dermal wound, and the rate of wound closure was assessed daily for 10 days, with adenosine receptor agonist CGS‐21680 as a positive control. Topical application of brassinosteroid significantly reduced wound size and accelerated wound healing in treated animals. mRNA levels of transforming growth factor beta and intercellular adhesion molecule 1 were significantly lower, while tumor necrosis factor alpha was nearly suppressed in the wounds from treated mice. Our data suggest that topical application of brassinosteroids accelerates wound healing by positively modulating inflammatory and reepithelialization phases of the wound repair process, in part by enhancing Akt signaling in the skin at the edges of the wound and enhancing migration of fibroblasts in the wounded area. Targeting this signaling pathway with brassinosteroids may represent a promising approach to the therapy of delayed wound healing.     

Siah2‐deficient mice show impaired skin wound repair

Hypoxia is associated with the dermal wound healing process and hypoxia signaling is presumed to be crucial for normal wound repair. The Siah2 ubiquitin ligase controls the abundance of hypoxia‐inducible factor‐1 alpha, and loss of Siah2 results in destabilization of hypoxia‐inducible factor‐1 alpha under hypoxia. Utilizing Siah2^?/? mice we demonstrate that cutaneous wound healing is impaired in these mice. Wounds in Siah2^?/? mice heal slower and are associated with delayed induction of myofibroblast infiltration and reduced collagen deposition. This coincides with delayed angiogenesis and reduced macrophage infiltration into the wounds of Siah2^?/? mice. We furthermore demonstrate that primary Siah2^?/? dermal fibroblasts have reduced migratory capacities and produce less collagen than wild‐type fibroblasts. Additionally, Siah2^?/? fibroblasts showed conserved responses to transforming growth factor‐β at the receptor level (pSmad 2C activation) but reduced responses downstream. Together, our data show, for the first time, that Siah2 is involved as a positive regulator in the wound healing response. Understanding the role of hypoxia signaling in tissue repair and fibrosis and interference with the hypoxia signaling pathway via regulation of Siah2 may provide new targets for clinical regulation of fibrosis and scarring.     

Angiopoietin-1 mediated effects on re-epithelialization: in vitro and in vivo analysis

Introduction: Adenoviral-mediated overexpression of Angiopoietin-1 (AdAng-1), surprisingly results in accelerated re-epithelialization. Because keratinocytes do not express Tie2, the Ang-1 receptor, we hypothesize that Ang-1 induces downstream growth factors. We employed an in vitro co-culture system and an excisional wound healing model in TGFα knock out (KO) and normal mice to examine Ang-1 wound healing effects. Methods: Fibroblasts transduced with AdAng-1, AdLacZ (MOI 20) or PBS are placed into co-culture with keratinocytes. Neutralizing antibodies (NAbs) to Ang-1, KGF, EGF, PDGF-B, PDGF-A, IGF-1, TGFα and TGFβ1 were added. Ki67 immunostaining of the keratinocytes was used to determine a proliferation index (PI). 6 mm crown wounds treated with 1 x 10⁸ PFU of AdAng-1 AdLacZ, or 20μl PBS were made in TGFα KO (n = 15) and control mice (n = 15). Wounds harvested at 5 days were analyzed for epithelial gap, granulation tissue formation, capillary density and KGF expression. Results: In co-culture, AdAng-1 results in increased keratinocyte PI compared to controls (Ang1 38.5% vs. LacZ 19% or PBS 16.3%). This response was completely inhibited by NAbs to Ang1 (17.1%) or TGFα (16%) and partially inhibited by NAbs to KGF (29.5%) or PDGF-B (25.6%). NAbs to PDGF-A, IGF-1, TGFβ or EGF had no effect. AdAng-1 treated wounds in TGFα KO mice showed significantly larger epithelial gaps compared to Ad-Ang-1 treated control mice (3.3 mm ± 0.1 vs. 2.7 mm ± 0.2, p < 0.01). However, in TGFα KO mice AdAng-1 treated wounds show significantly reduced epithelial gaps (Ang-1 3.3 mm ±.1, LacZ 4.4±.2, PBS 4.4±.3, p<.001) and increased vessel density compared to controls (Ang1 11.1 Caps/HPF, LacZ 4.1 ± 0.3, PBS 4.4 ± 0.3, p < 0.0001). KGF immunostaining localized to the advancing epithelial margin, was markedly enhanced in AdAng-1 treated wounds in both TGFα KO and control mice. Conclusions: The effects of Ang-1 on keratinocyte proliferation are mediated indirectly by TGFα, and KGF. In vivo, TGFα is an important mediator of Ang-1 effect on reepithelialization, but Ang-1 also enhances KGF expression. These results suggest that wound healing effects of Ang-1 over expression are to due to dermal epidermal signaling mediated by TGFα and KGF.     

Hyperbaric oxygen therapy activates hypoxia‐inducible factor 1 (HIF‐1), which contributes to improved wound healing in diabetic mice

Hyperbaric oxygen (HBO) therapy has been used as an adjunctive therapy for diabetic foot ulcers, although its mechanism of action is not completely understood. Recently, it has been shown that HBO mobilizes the endothelial progenitor cells (EPCs) from bone marrow that eventually will aggregate in the wound. However, the gathering of the EPCs in diabetic wounds is impaired because of the decreased levels of local stromal‐derived factor‐1α (SDF‐1α). Therefore, we investigated the influence of HBO on hypoxia‐inducible factor 1 (HIF‐1), which is a central regulator of SDF‐1α and is down‐regulated in diabetic wounds. The effects of HBO on HIF‐1α function were studied in human dermal fibroblasts, SKRC7 cells, and HIF‐1α knock‐out and wild‐type mouse embryonic fibroblasts using appropriate techniques (Western blot, quantitative polymerase chain reaction, and luciferase hypoxia‐responsive element reporter assay). Cellular proliferation was assessed using H³‐thymidine incorporation assay. The effect of HIF in combination with HBOT was tested by inoculating stable HIF‐1α‐expressing adenovirus (Adv‐HIF) into experimental wounds in db/db mice exposed to HBO. HBO activates HIF‐1α at several levels by increasing both HIF‐1α stability (by a non‐canonical mechanism) and activity (as shown both by induction of relevant target genes and by a specific reporter assay). HIF‐1α induction has important biological relevance because the induction of fibroblast proliferation in HBO disappears when HIF‐1α is knocked down. Moreover, the local transfer of stable HIF‐1α‐expressing adenovirus (Adv‐HIF) into experimental wounds in diabetic (db/db mice) animals has an additive effect on HBO‐mediated improvements in wound healing. In conclusion, HBO stabilizes and activates HIF‐1, which contributes to increased cellular proliferation. In diabetic animals, the local transfer of active HIF further improves the effects of HBO on wound healing.     

Nucleolin enhances the proliferation and migration of heat‐denatured human dermal fibroblasts

Denatured dermis, a part of dermis in burned skin, has the ability to restore its normal morphology and functions after their surrounding microenvironment is improved. However, the cellular and molecular mechanisms by which the denatured dermis could improve wound healing are still unclear. This study aimed to investigate the role of nucleolin during the recovery of heat‐denatured human dermal fibroblasts. Nucleolin mRNA and protein expression were significantly increased time‐dependently during the recovery of heat‐denatured human dermal fibroblasts (52 °C, 30 seconds). Heat‐denaturation promoted a time‐dependent cell proliferation, migration, chemotaxis, and scratched wound healing during the recovery of human dermal fibroblasts. These effects were prevented by knockdown of nucleolin expression with small interference RNA (siRNA), whereas overexpression of nucleolin enhanced cell proliferation, migration, and chemotaxis of human dermal fibroblasts with heat‐denaturation. In addition, the expression of transforming growth factor‐beta 1(TGF‐β1) was significantly increased during the recovery of heat‐denatured dermis and human dermal fibroblasts. TGF‐β1 expression was up‐regulated by nucleolin in human dermal fibroblasts. The results suggest that nucleolin expression is up‐regulated, and play an important role in promoting cell proliferation, migration, and chemotaxis of human dermal fibroblasts during the recovery of heat‐denatured dermis with a mechanism probably related to TGF‐β1.     

Ephrin-A2 affects wound healing and scarring in a murine model of excisional injury

Ephrin ligand/Eph receptor signaling is important in both tissue development and homeostasis. There is increasing evidence that Ephrin/Eph signaling is important in the skin, involved in hair follicle cycling, epidermal differentiation, cutaneous innervation and skin cancer. However, there is currently limited information on the role of Ephrin/Eph signaling in cutaneous wound healing. Here we report the effects of the Ephrin-A2 and A5 ligands on wound healing. Using Ephrin-A2^−/−, Ephrin-A5^−/− and Ephrin-A2A5^−/− transgenic mice, in vitro wound healing assays were conducted using isolated keratinocytes and fibroblasts. Ephrin-A2^−/−, Ephrin-A2A5^−/− and wild type mice with excisional wounds were used to analyze the impact of these ligands on wound closure, scar outcome, collagen orientation and re-innervation in vivo.The absence of the Ephrin-A2 and A5 ligands did not have any effect on dermal fibroblast proliferation or on fibroblast or keratinocyte migration. The loss of Ephrin-A2 and A5 ligands did not impact on the rate of wound closure or re-innervation after injury. However, changes in the gross morphology of the healed scar and in collagen histology of the scar dermis were observed in transgenic mice. Therefore Ephrin-A2 and A5 ligands may play an important role in final scar appearance associated with collagen deposition and structure.     

Matrix metalloproteinase inhibition delays wound healing and blocks the latent transforming growth factor‐β1‐promoted myofibroblast formation and function

The ability to regulate wound contraction is critical for wound healing as well as for pathological contractures. Matrix metalloproteinases (MMPs) have been demonstrated to be obligatory for normal wound healing. This study examined the effect that the broad‐spectrum MMP inhibitor BB‐94 has when applied topically to full‐thickness skin excisional wounds in rats and its ability to inhibit the promotion of myofibroblast formation and function by the latent transforming‐growth factor‐β1 (TGF‐β1). BB‐94 delayed wound contraction, as well as all other associated aspects of wound healing examined, including myofibroblast formation, stromal cell proliferation, blood vessel formation, and epithelial wound coverage. Interestingly, BB‐94 dramatically increased the level of latent and active MMP‐9. The increased levels of active MMP‐9 may eventually overcome the ability of BB‐94 to inhibit this MMP and may explain why wound contraction and other associated events of wound healing were only delayed and not completely inhibited. BB‐94 was also found to inhibit the ability of latent TGF‐β1 to promote the formation and function of myofibroblasts. These results suggest that BB‐94 could delay wound closure through a twofold mechanism; by blocking keratinocyte migration and thereby blocking the necessary keratinocyte–fibroblast interactions needed for myofibroblast formation and by inhibiting the activation of latent TGF‐β1.     

A pre‐clinical functional assessment of an acellular scaffold intended for the treatment of hard‐to‐heal wounds

The majority of the population experience successful wound‐healing outcomes; however, 1–3% of those aged over 65 years experience delayed wound healing and wound perpetuation. These hard‐to‐heal wounds contain degraded and dysfunctional extracellular matrix (ECM); yet, the integrity of this structure is critical in the processes of normal wound healing. Here, we evaluated a novel synthetic matrix protein for its ability to act as an acellular scaffold that could replace dysfunctional ECM. In this regard, the synthetic protein was subjected to adsorption and diffusion assays using collagen and human dermal tissues; evaluated for its ability to influence keratinocyte and fibroblast attachment, migration and proliferation and assessed for its ability to influence in vivo wound healing in a porcine model. Critically, these experiments demonstrate that the matrix protein adsorbed to collagen and human dermal tissue but did not diffuse through human dermal tissue within a 24‐hour observation period, and facilitated cell attachment, migration and proliferation. In a porcine wound‐healing model, significantly smaller wound areas were observed in the test group compared with the control group following the third treatment. These data provide evidence that the synthetic matrix protein has the ability to function as an acellular scaffold for wound‐healing purposes.     

Lysophosphatidic acid stimulates epidermal growth factor‐family ectodomain shedding and paracrine signaling from human lung fibroblasts

Lysophospatidic acid (LPA) is a bioactive lipid mediator implicated in tissue repair and wound healing. It mediates diverse functional effects in fibroblasts, including proliferation, migration and contraction, but less is known about its ability to evoke paracrine signaling to other cell types involved in wound healing. We hypothesized that human pulmonary fibroblasts stimulated by LPA would exhibit ectodomain shedding of epidermal growth factor receptor (EGFR) ligands that signal to lung epithelial cells. To test this hypothesis, we used alkaline phosphatase‐tagged EGFR ligand plasmids transfected into lung fibroblasts, and enzyme‐linked immunosorbent assays to detect shedding of native ligands. LPA induced shedding of alkaline phosphatase‐tagged heparin‐binding epidermal growth factor (HB‐EGF), amphiregulin, and transforming growth factor‐a; non‐transfected fibroblasts shed amphiregulin and HBEGF under baseline conditions, and increased shedding of HB‐EGF in response to LPA. Treatment of fibroblasts with LPA resulted in elevated phosphorylation of extracellular signal‐regulated kinase 1/2, enhanced expression of mRNA for c‐fos, HB‐EGF and amphiregulin, and enhanced proliferation at 96 hours. However, none of these fibroblast responses to LPA required ectodomain shedding or EGFR activity. To test the ability of LPA to stimulate paracrine signaling from fibroblasts, we transferred conditioned medium from LPA‐stimulated cells, and found enhanced EGFR and extracellular signal‐regulated kinase 1/2 phosphorylation in reporter A549 cells in excess of what could be accounted for by transferred LPA alone. These data show that LPA mediates EGF‐family ectodomain shedding, resulting in enhanced paracrine signaling from lung fibroblasts to epithelial cells.     

Transgenic mice overexpressing CD109 in the epidermis display decreased inflammation and granulation tissue and improved collagen architecture during wound healing

Transforming growth factor‐β (TGF‐β) is a multifunctional growth factor involved in all aspects of wound healing. TGF‐β accelerates wound healing, but an excess of its presence at the wound site has been implicated in pathological scar formation. Our group has recently identified CD109, a glycophosphatidylinositol‐anchored protein, as a novel TGF‐β coreceptor and inhibitor of TGF‐β signaling in vitro. To determine the effects of CD109 in vivo on wound healing, we generated transgenic mice overexpressing CD109 in the epidermis. In excisional wounds, we show that CD109 transgenic mice display markedly reduced macrophage and neutrophil recruitment, granulation tissue area, and decreased Smad2 and Smad3 phosphorylation, whereas wound closure remains unaffected as compared with wild‐type littermates. Futhermore, we demonstrate that the expression of the proinflammatory cytokines interleukin‐1α and monocyte chemoattractant protein‐1, and extracellular matrix components is markedly decreased during wound healing in CD109 transgenic mice. In incisional wounds, CD109 transgenic mice show improved dermal architecture, whereas the tensile strength of the wound remains unchanged. Taken together, our findings demonstrate that CD109 overexpression in the epidermis reduces inflammation and granulation tissue area and improves collagen organization in vivo.     

Inhibited proliferation of fibroblasts derived from chronic diabetic wounds and normal dermal fibroblasts treated with high glucose is associated with increased formation of L-lactate

Diabetes is accompanied by delayed wound healing and insufficient granulation tissue formation, possibly because of a defect in fibroblast function. We have previously shown that fibroblasts derived from chronic diabetic foot ulcers have lower proliferation compared with those from uninjured skin. The aim of this study was to investigate possible mechanisms explaining the impaired fibroblast proliferation observed in fibroblasts from non-insulin-dependent diabetes mellitus chronic wounds and normal fibroblasts cultured in high glucose. Fibroblasts from two groups of patients were studied: nondiabetic patients with chronic venous stasis ulcers and non-insulin-dependent diabetes mellitus patients with chronic diabetic wounds. Biopsies from both uninjured skin and wounds were taken from the same patients to serve as sources of fibroblasts. A fluorometric method was used to determine DNA content, and a spectrophotometric lactate oxidase method was used for lactate level analysis. We found a dose-dependent inhibition of normal fibroblast proliferation when adding conditioned media from non-insulin-dependent diabetes mellitus wound fibroblasts. The conditioned medium, from these cells showed elevated L-lactate levels, 6.3 ± 0.7 mmol/L, compared with media derived from nondiabetic, 2.1 ± 0.3 mmol/L (p < 0.01), and diabetic uninjured skin fibroblasts, 3.5 ± 0.6 mmol/L, and from chronic nondiabetic wound fibroblasts 2.9 ± 0.3 mmol/L. Addition of 6 mmol/L L-lactate to uninjured normal fibroblasts resulted in decreased DNA content (58 ± 7%, p < 0.01). Previously we have shown that high glucose concentrations inhibit fibroblast proliferation and induce growth factor resistance. When increasing the amount of D-glucose in the media, L-lactate levels increased in all cell types. When the uninjured normal cells were treated with β-hydroxybutyrate, the total DNA content decreased by 42 ± 5% (p < 0.05), with no significant increase in the L-lactate levels. These observations indicate that L-lactate production may be of importance for fibroblast proliferation in vitro and may play a role in fibroblast proliferation in vivo.     

Wound healing in Mac‐1 deficient mice

Mac‐1 (CD11b/CD18) is a macrophage receptor that plays several critical roles in macrophage recruitment and activation. Because macrophages are essential for proper wound healing, the impact of Mac‐1 deficiency on wound healing is of significant interest. Prior studies have shown that Mac‐1^?/? mice exhibit deficits in healing, including delayed wound closure in scalp and ear wounds. This study examined whether Mac‐1 deficiency influences wound healing in small excisional and incisional skin wounds. Three millimeter diameter full thickness excisional wounds and incisional wounds were prepared on the dorsal skin of Mac‐1 deficient (Mac‐1^?/?) and wild type (WT) mice, and wound healing outcomes were examined. Mac‐1 deficient mice exhibited a normal rate of wound closure, generally normal levels of total collagen, and nearly normal synthesis and distribution of collagens I and III. In incisional wounds, wound breaking strength was similar for Mac‐1^?/? and WT mice. Wounds of Mac‐1 deficient mice displayed normal total macrophage content, although macrophage phenotype markers were skewed as compared to WT. Interestingly, amounts of TGF‐β1 and its downstream signaling molecules, SMAD2 and SMAD3, were significantly decreased in the wounds of Mac‐1 deficient mice compared to WT. The results suggest that Mac‐1 deficiency has little impact on the healing of small excisional and incisional wounds. Moreover, the findings demonstrate that the effect of single genetic deficiencies on wound healing may markedly differ among wound models. These conclusions have implications for the interpretation of the many prior studies that utilize a single model system to examine wound healing outcomes in genetically deficient mice.     

Differential effects of planktonic and biofilm MRSA on human fibroblasts

Bacteria colonizing chronic wounds often exist as biofilms, yet their role in chronic wound pathogenesis remains unclear. Staphylococcus aureus biofilms induce apoptosis in dermal keratinocytes, and given that chronic wound biofilms also colonize dermal tissue, it is important to investigate the effects of bacterial biofilms on dermal fibroblasts. The effects of a predominant wound pathogen, methicillin‐resistant S. aureus, on normal, human, dermal fibroblasts were examined in vitro. Cell‐culture medium was conditioned with equivalent numbers of either planktonic or biofilm methicillin‐resistant S. aureus and then fed to fibroblast cultures. Fibroblast response was evaluated using scratch, viability, and apoptosis assays. The results suggested that fibroblasts experience the same fate when exposed to the soluble products of either planktonic or biofilm methicillin‐resistant S. aureus, namely limited migration followed by death. Enzyme‐linked immunosorbent assays demonstrated that fibroblast production of cytokines, growth factors, and proteases were differentially affected by planktonic and biofilm‐conditioned medium. Planktonic‐conditioned medium induced more interleukin‐6, interleukin‐8, vascular endothelial growth factor, transforming growth factor‐β1, heparin‐bound epidermal growth factor, matrix metalloproteinase‐1, and metalloproteinase‐3 production in fibroblasts than the biofilm‐conditioned medium. Biofilm‐conditioned medium induced more tumor necrosis factor‐α production in fibroblasts compared with planktonic‐conditioned medium, and suppressed metalloproteinase‐3 production compared with controls.     

TGFβ Inducible Early Gene‐1 Plays an Important Role in Mediating Estrogen Signaling in the Skeleton

TGFβ Inducible Early Gene‐1 (TIEG1) knockout (KO) mice display a sex‐specific osteopenic phenotype characterized by low bone mineral density, bone mineral content, and overall loss of bone strength in female mice. We, therefore, speculated that loss of TIEG1 expression would impair the actions of estrogen on bone in female mice. To test this hypothesis, we employed an ovariectomy (OVX) and estrogen replacement model system to comprehensively analyze the role of TIEG1 in mediating estrogen signaling in bone at the tissue, cell, and biochemical level. Dual‐energy X‐ray absorptiometry (DXA), peripheral quantitative computed tomography (pQCT), and micro‐CT analyses revealed that loss of TIEG1 expression diminished the effects of estrogen throughout the skeleton and within multiple bone compartments. Estrogen exposure also led to reductions in bone formation rates and mineralizing perimeter in wild‐type mice with little to no effects on these parameters in TIEG1 KO mice. Osteoclast perimeter per bone perimeter and resorptive activity as determined by serum levels of CTX‐1 were differentially regulated after estrogen treatment in TIEG1 KO mice compared with wild‐type littermates. No significant differences were detected in serum levels of P1NP between wild‐type and TIEG1 KO mice. Taken together, these data implicate an important role for TIEG1 in mediating estrogen signaling throughout the mouse skeleton and suggest that defects in this pathway are likely to contribute to the sex‐specific osteopenic phenotype observed in female TIEG1 KO mice. © 2014 American Society for Bone and Mineral Research.     

Molecular events underlying maggot extract promoted rat in vivo and human in vitro skin wound healing

Maggot extracts promote wound healing, but their bioactive part(s) and molecular effects on the regenerating tissues/cells remain largely unclear. These issues are addressed here by treating rat skin wounds, human keratinocyte line/HaCat and fibroblasts with maggot secretion/excretion, and the extracts of maggots without and with secretion/excretion. The wound closure rates, cell proliferation activities, and statuses of wound healing‐related signaling pathways (STAT3, Notch1, Wnt2, NF‐κB, and TGF‐beta/Smad3) and their downstream gene expression (c‐Myc, cyclin D1, and VEGF) are evaluated by multiple approaches. The results reveal that the maggot extracts, especially the one from the maggots without secretion/excretion, show the best wound healing‐promoting effects in terms of quicker wound closure rates and more rapid growth of keratinocytes and fibroblasts. Of the five signaling pathways checked, the ones mediated by TGF‐beta/Smad3, and STAT3 are activated in the untreated wounds and become further enhanced by the maggot extracts, accompanied with c‐Myc, VEGF, and cyclin D1 up‐regulation. Our results thus show (1) that both body extract and secretion/excretion of maggots contain favorable wound healing elements and (2) that the enhancement of TGF‐beta/Smad3 and STAT3 signaling activities may be the main molecular effects of maggot extracts on the wound tissues.     

Hepatitis C Virus Infection is Highly Correlated with Hepatocellular Apoptosis and Transforming Growth Factor‐β1 MRNA Expression in Patients with Chronic Hepatitis C

Introduction:  The cellular phases (granulation, reepithelialization, and dermal remodelling) of the healing process involve many cell types. Fibroblasts and myofibroblasts are the key cells in granulation tissue formation and wound contraction.
Objective:  To compare the effects on cultured human fibroblasts of a new nonadhesive lipidocolloid wound dressing, Urgotul^®, with five other wound dressings including impregnated gauzes and some other modern wound dressings.
Method:  Cultures in monolayer were used to study the morphology and growth of fibroblasts. The Bell model of cultured dermis equivalents was used to investigate myofibroblast differentiation. These cultures were labelled α‐SM actin and F‐actin.
Results:  Two of the tested dressings induced cytotoxic effects on the fibroblasts. They were found to inhibit cell growth (greater than 60%) and to disturb cell shape and cytoskeletal differentiation. Urgotul^® and the remaining three dressings showed no effect on proliferation. However some of them modified fibroblast morphology and affected F‐actin distribution.
Conclusion:  Depending on their nature and components, wound dressings may respect or affect in vitro fibroblast behaviour (proliferation, morphology, and α‐SM actin and F‐actin distribution). The observed in vitro findings require further investigations.