Effects of Human Adipose-derived Stem Cells on Cutaneous Wound Healing in Nude Mice

Background

Despite numerous treatments available for deteriorated cutaneous wound healing such as a diabetic foot, there is still the need for more effective therapy. Adipose-derived stem cells (ASCs) are mesenchymal stem cells, which are self-renewing and multipotent. Mesenchymal stem cells have the potential for tissue repair and regeneration.

Objective

To investigate the effects of human ASCs on the healing of cutaneous wounds in nude mice.

Methods

15-mm round full-thickness skin defects were generated on the back of BALB/c nude mice. The mice were divided into three groups for wound coverage: (i) human ASCs-populated collagen gel, (ii) human dermal fibroblasts-populated collagen gel, and (iii) collagen gel alone. Wound contraction was prevented with a splint method. Wound size was measured 10 days after injury. At 28 days histological analysis was performed.

Results

Both ASCs and dermal fibroblasts accelerated wound closure, but dermal fibroblasts were more effective than ASCs. At 28 days, the dermal portion of ASCs or dermal fibroblasts wound scars were thicker than collagen gel wound scars.

Conclusion

ASCs and dermal fibroblasts stimulate cutaneous wound healing and improve scar thickness.     

COMP-angiopoietin 1 Gene Transfer Enhances Cutaneous Wound Healing by Promoting Angiogenesis

Background

Angiogenesis is crucial for wound healing and exogenous supplements of the angiogenic growth factors have been known to promote cutaneous wound healing. Angiopoietin (Ang) 1 is a recently discovered angiogenic factor and there have been few studies of its effect on cutaneous wound healing.

Objective

We examined the effect of Ang 1 on cutaneous wound healing.

Methods

Cartilage oligomeric matrix protein (COMP)-Ang 1 (Ade-COMP-Ang 1)- was intravenously injected to rats two days before surgery creating full-thickness wounds. The clinical wound healing rate and the number of vessels in the skin samples were evaluated on days 3, 7 and 14 post operation.

Results

At post-operation day 3, 7 and 14, the clinical wound healing rate was 38.3%, 59.4% and 92.1%, respectively, in the Ade-COMP-Ang 1-treated group, compared with 20.5%, 47.5% and 87.3%, respectively, in the Ade-LacZ-treated group. There were significant differences in the results of day 3 and day 7 between two groups (p<0.05). Histopathologically, the number of the vessels of the Ade-COMP-Ang 1-treated group was 73.7, 94.1 and 62.7 at day 3, 7 and 14, compared with that of the Ade-LacZ-treated group, 53.5, 83.9, and 56.9. The differences in the results of the two groups were statistically significant (p<0.05).

Conclusion

These results indicate that Ade-COMP-Ang 1 therapy significantly accelerats wound healing by promoting angiogenesis. However, further study using Ade-COMP-Ang 1 gene therapy for chronic wounds in which the formation of new blood vessels is impaired is needed in the near future.     

Stimulation of the Extracellular Matrix Production in Dermal Fibroblasts by Velvet Antler Extract

Background

Fibroblasts produce many components of the extracellular matrix (ECM) and so they contribute to the maintenance of connective tissue integrity.

Objective

The aim of this study is to evaluate the effect of velvet antler extract (VAE) on the ECM production of dermal fibroblasts cultured in vitro.

Methods

Primary cultured human dermal fibroblasts were treated with VAE, and then the ECM production was determined by RT-PCR, ELISA and Western blot analysis. Furthermore, the change of gene expression according to VAE treatment was evaluated by cDNA microarray.

Results

VAE accelerated the growth of fibroblasts in a dose-dependent manner. VAE increased the production of several ECM components, including type 1 collagen, fibronectin and elastin. In line with these results, the phosphorylations of p42/44 ERK and p38 mitogen-activated protein kinase were markedly increased by VAE, suggesting that the enhancement of ECM production may be linked to the activation of intracellular signaling cascades. VAE also significantly increased cell migration on an in vitro scratch wound test. In cDNA microarray, many genes related with connective tissue integrity were identified to be up-regulated by VAE.

Conclusion

These results suggest that VAE has a potential to stimulate ECM production, and VAE may be applicable for maintaining the skin''s texture.     

A Novel Compound Rasatiol Isolated from Raphanus sativus Has a Potential to Enhance Extracellular Matrix Synthesis in Dermal Fibroblasts

Background

The fibrous proteins of extracellular matrix (ECM) produced by dermal fibroblast contributes to the maintenance of connective tissue integrity.

Objective

This study is carried out to identify the bioactive ingredient from natural products that enhances ECM production in dermal fibroblasts.

Methods

Bioassay-directed fractionation was used to isolate the active ingredient from natural extracts. The effects of rasatiol (isolated from Raphanus sativus) on ECM production in primary cultured human dermal fibroblasts was investigated by enzyme linked immunosorbent assay and western blot analysis.

Results

Rasatiol accelerated fibroblast growth in a dose-dependent manner and increased the production of type 1 collagen, fibronectin and elastin. Phosphorylation of p42/44 extracellular signal-regulated kinase, p38 mitogen-activated protein kinase, and Akt was remarkably increased by rasatiol, indicating that enhanced ECM production is linked to the activation of intracellular signaling cascades.

Conclusion

These results indicate that rasatiol stimulates the fibrous components of ECM production, and may be applied to the maintenance of skin texture.     

Interleukin-17 and Interleukin-22 Induced Proinflammatory Cytokine Production in Keratinocytes via Inhibitor of Nuclear Factor ��B Kinase-�� Expression

Background

The pathogenesis of psoriasis may involve the interleukin (IL)-23 and Th17-mediated immune responses. Th17 cells secret IL-17 and IL-22, which mediates dermal inflammation and acanthosis.

Objective

As inhibitor of nuclear factor κB kinase-α (IKKα) has been previously identified as a primary regulator of keratinocyte differentiation and proliferation, we proposed that IL-17 and IL-22 might affect keratinocyte differentiation by changing the expression of IKKα.

Methods

We employed HaCaT cells maintained culture medium at a low calcium concentration (0.06 mM) and induced differentiation by switching to the high concentration (2.8 mM) media with IL-17 or IL-22, then compared the IKKα expression and the cell cycle. We employed reconstituted human epidermal skin (Neoderm) and mice ears for the in vivo studies.

Results

Elevated calcium concentration induced IKKα expression and terminal differentiation with cell cycle arrest in HaCaT cell cultures. Moreover, IL-17 and IL-22 treatment also induced IKKα in HaCaT cells and reconstituted human epidermis. IKKα induction was also noted, following the injection of IL-17 and IL-22 into mice ears.

Conclusion

Although the induction of IKKα was accompanied by keratinocyte differentiation, IL-17 and IL-22 did not affect calcium-mediated differentiation or the cell cycle. Rather, IL-17 and IL-22 appear to contribute to the inflammation occurring via the induction of IKKα from keratinocytes or skin layers.     

Synergistic Effect of Bone Marrow-Derived Mesenchymal Stem Cells and Platelet-Rich Plasma in Streptozotocin-Induced Diabetic Rats

Background

Diabetic wounds are a major clinical challenge, because minor skin wounds can lead to chronic, unhealed ulcers and ultimately result in infection, gangrene, or even amputation. Studies on bone marrow derived mesenchymal stem cells (BMSCs) and a series of growth factors have revealed their many benefits for wound healing and regeneration. Platelet-rich plasma (PRP) may improve the environment for BMSC development and differentiation. However, whether combined use of BMSCs and PRP may be more effective for accelerating diabetic ulcer healing remains unclear.

Objective

We investigated the efficacy of BMSCs and PRP for the repair of refractory wound healing in a diabetic rat model.

Methods

Forty-eight rats with diabetes mellitus induced by streptozotocin were divided into four groups: treatment with BMSCs plus PRP, BMSCs alone, PRP alone, phosphate buffered saline. The rate of wound closure was quantified. A histopathological study was conducted regarding wound depth and the skin edge at 7, 14, and 28 days after surgery.

Results

Wound healing rates were significantly higher in the BMSC plus PRP group than in the other groups. The immunohistochemistry results showed that the expression of platelet/endothelial cell adhesion molecule 1, proliferating cell nuclear antigen, and transforming growth factor-β1 increased significantly in the BMSC plus PRP group compared to the other treatment groups. On day 7, CD68 expression increased significantly in the wounds of the BMSC plus PRP group, but decreased markedly at day 14 compared to the controls.

Conclusion

The combination of BMSCs and PRP aids diabetic wound repair and regeneration.     

The Effects of 830 nm Light-Emitting Diode Therapy on Acute Herpes Zoster Ophthalmicus: A Pilot Study

Background

Skin lesions and pain are the most distinctive features of herpes zoster. Light-emitting diode (LED) therapy is an effective treatment known for its wound-healing effects.

Objective

To determine whether the LED treatment affects wound healing and acute pain in acute herpes zoster ophthalmicus.

Methods

We recruited 28 consecutive Korean patients with acute herpes zoster ophthalmicus for the study. In the control group (group A), 14 subjects received oral famcyclovir. In the experimental group (group B), 14 subjects received oral famcyclovir and 830 nm LED phototherapy on days 0, 4, 7, and 10. In order to estimate the time for wound healing, we measured the duration from the vesicle formation to when the lesion crust fell off. The visual analogue scale (VAS) was used for the estimation of pain on days 4, 7, 10, and 14.

Results

The mean time required for wound healing was 13.14±2.34 days in group B and 15.92±2.55 days in group A (p=0.006). From day 4, the mean VAS score showed a greater improvement in group B, compared with group A. A marginal but not statistically significant difference in the VAS scores was observed between the two groups (p=0.095).

Conclusion

LED treatment for acute herpes zoster ophthalmicus leads to faster wound healing and a lower pain score.     

Cholesterol, a Major Component of Caveolae, Down-regulates Matrix Metalloproteinase-1 Expression through ERK/JNK Pathway in Cultured Human Dermal Fibroblasts

Background

Cholesterol is a major component of specialized membrane microdomains known as lipid rafts or caveolae, which modulate the fluidity of biological membranes. Membrane cholesterol therefore plays an important role in cell signaling and vesicular transport.

Objective

In this study, we investigated the effects of cholesterol on matrix metalloproteinase-1 (MMP-1) expression in human dermal fibroblasts.

Methods

MMP-1 mRNA and protein expression were determined by RT-PCR and Western blotting, respectively. AP-1 DNA binding activity was detected by electrophoretic mobility shift assays. The amount of cholesterol was analyzed by cholesterol assay kit.

Results

We observed that MMP-1 mRNA and protein expression was dose-dependently decreased by cholesterol treatment. In contrast, cholesterol depletion by a cholesterol depletion agent, methyl-beta-cyclodextrin (MβCD) in human dermal fibroblasts, increased MMP-1 mRNA and protein expression in a dose-dependent manner. Also, we investigated the regulatory mechanism of MβCD-induced MMP-1 expression: cholesterol depletion by MβCD, activated ERK1/2 and JNK, but not p38 MAPK cascade, and it also significantly increased c-Jun phosphorylation, c-Fos expression and activator protein-1 binding activity. Furthermore, the inhibition of ERK or JNK with specific chemical inhibitors prevented MβCD-induced MMP-1 expression, which indicates that ERK and JNK play an important role in cholesterol depletion-mediated MMP-1 induction. In addition, MβCD-induced phosphorylation of ERK and JNK and MMP-1 expression were suppressed by cholesterol repletion.

Conclusion

Our results suggest that cholesterol regulates MMP-1 expression through the control of ERK and JNK activity in human dermal fibroblasts.     

Scalded Skin of Rat Treated by Using Fibrin Glue Combined with Allogeneic Bone Marrow Mesenchymal Stem Cells

Background

It is difficult to achieve satisfactory results with the traditional treatment of large-area skin defects and deep burns.

Objective

To test the treatment effect of an active dressing film made of a mixture of fibrin glue and bone marrow mesenchymal stem cells (BMSCs) for repairing burn wounds on the skin of rats.

Methods

Two scald wounds were made on the back of each rat. A total of 30 scald wounds were randomly divided into 3 groups, with 10 wounds in each group. In the experimental treatment group, the scald wounds were covered with the fibrin glue and BMSC mixture. The wounds of the experimental control group were covered with fibrin glue only. No intervention was administered to the blank control group. Thirty days after treatment, pathological sections were cut from the scalded local tissues of all rats from the 3 groups and observed with a microscope.

Results

The speed of scald wound healing in the experimental treatment group was faster than the other 2 groups. In the experimental treatment group, histopathological analysis revealed that the sebaceous glands showed obviously proliferous at the edge of the new tissue and gradually extended to the deep dermal layer of the new tissue.

Conclusion

BMSCs may have an active role in promoting skin tissue repair and generating skin appendages. Allogeneic BMSCs mixed with fibrin glue can contribute to the quick formation of a film-like gel over the scald wounds, which might be of significance for emergency treatment and skin-grafting operations.     

Phototherapy promotes healing of cutaneous wounds in undernourished rats

BACKGROUND

Various studies have shown that phototherapy promotes the healing of cutaneous wounds.

OBJECTIVE

To investigate the effect of phototherapy on healing of cutaneous wounds in nourished and undernourished rats.

METHODS

Forty rats, 20 nourished plus 20 others rendered marasmus with undernourishment, were assigned to four equal groups: nourished sham, nourished Light Emitting Diode treated, undernourished sham and undernourished Light Emitting Diode treated. In the two treated groups, two 8-mm punch wounds made on the dorsum of each rat were irradiated three times per week with 3 J/cm² sq cm of combined 660 and 890nm light; wounds in the other groups were not irradiated. Wounds were evaluated with digital photography and image analysis, either on day 7 or day 14, with biopsies obtained on day 14 for histological studies.

RESULTS

Undernourishment retarded the mean healing rate of the undernourished sham wounds (p < 0.01), but not the undernourished Light emission diode treated wounds, which healed significantly faster (p < 0.001) and as fast as the two nourished groups. Histological analysis showed a smaller percentage of collagen in the undernourished sham group compared with the three other groups, thus confirming our photographic image analysis data.

CONCLUSION

Phototherapy reverses the adverse healing effects of undernourishment. Similar beneficial effects may be achieved in patients with poor nutritional status.     

Cold atmospheric plasma (CAP) activates angiogenesis-related molecules in skin keratinocytes,fibroblasts and endothelial cells and improves wound angiogenesis in an autocrine and paracrine mode

Background

Cold atmospheric plasma (CAP) emerged as a novel therapeutic field with applications developed for bacterial sterilization, wound healing and cancer treatment. For clinical implementation it is important to know how CAP works and which molecular changes occur after the CAP treatment. Vascularization is an important step during wound healing, however, the effects of CAP on wound angiogenesis are not well examined so far. Furthermore, it has not been investigated, whether CAP primarily affects endothelial cells directly or via paracrine mechanisms to modulate the vasculature.

Angiogenic effects of cryosurgery with liquid nitrogen on the normal skin of rats,through morphometric study

BACKGROUND

Cryosurgery is an efficient therapeutic technique used to treat benign and malignant cutaneous diseases. The primary active mechanism of cryosurgery is related to vascular effects on treated tissue. After a cryosurgical procedure, exuberant granulation tissue is formed at the injection site, probably as a result of angiogenic stimulation of the cryogen and inflammatory response, particularly in endothelial cells.

OBJECTIVE

To evaluate the angiogenic effects of freezing, as part of the phenomenon of healing rat skin subjected to previous injury.

METHODS

Two incisions were made in each of the twenty rats, which were divided randomly into two groups of ten. After 3 days, cryosurgery with liquid nitrogen was performed in one of incisions. The rats'' samples were then collected, cut and stained to conduct histopathological examination, to assess the local angiogenesis in differing moments and situations.

RESULTS

It was possible to demonstrate that cryosurgery, in spite of promoting cell death and accentuated local inflammation soon after its application, induces quicker cell proliferation in the affected tissue and maintenance of this rate in a second phase, than in tissue healing without this procedure.

CONCLUSIONS

These findings, together with the knowledge that there is a direct relationship between mononuclear cells and neovascularization (the development of a rich system of new vessels in injury caused by cold), suggest that cryosurgery possesses angiogenic stimulus, even though complete healing takes longer to occur. The significance level for statistical tests was 5% (p<0,05).     

Melanoma inhibitory activity in Brazilian patients with cutaneous melanoma

BACKGROUND:

Melanoma inhibitory activity is a protein secreted by melanoma cells and has been used as a tumor marker. Increased Melanoma inhibitory activity serum levels are related to metastatic disease or tumor recurrence. Currently there are no studies on Melanoma inhibitory activity and cutaneous melanoma involving Brazilian patients.

OBJECTIVE:

To evaluate the performance and feasibility of measuring Melanoma inhibitory activity levels in Brazilian patients with cutaneous melanoma.

METHODS:

Blood was obtained from ten patients with proved metastatic cutaneous melanoma (Group 1), 15 patients resected for cutaneous melanoma without metastasis (Group 2) and 5 healthy donors (Group 3). Melanoma inhibitory activity was measured using a commercially available ELISA kit.

RESULTS:

There was a statistically significant difference of Melanoma inhibitory activity levels between patients with and without metastasis (p=0.002), and between patients with metastasis and healthy donors (p=0.002). There was no difference between patients without metastasis and healthy donors (p=0.443).

CONCLUSION:

Melanoma inhibitory activity is a tumor marker for cutaneous melanoma and the Melanoma inhibitory activity-ELISA test can be easily performed. Patients with metastasis have increased Melanoma inhibitory activity serum levels when compared to patients without metastasis and healthy donors.     

Tissue immunostaining for factor XIIIa in dermal dendrocytes of pityriasis alba skin lesions

BACKGROUND

Pityriasis alba affects 1% of the world population and about 9.9% of the children in Brazil. However, its etiology remains uncertain.

OBJECTIVE

The objective of the present study was to evaluate the immunoexpression of factor XIIIa in dermal dendrocytes of skin lesions of pityriasis alba.

METHOD

Twenty patients with pityriasis alba and 20 patients with atopic dermatitis underwent biopsy. The dermal dendrocytes marked by factor XIIIa were counted by means of immunohistochemical analysis.

RESULTS

The mean amount of dermal dendrocytes found in the patients with pityriasis alba was 2, whereas in the patients with atopic dermatitis it was 4, with a statistically significant difference between them. A cutoff point of 3 cells/square inch was established to differentiate pityriasis alba from atopic dermatitis, with 80% sensibility and 90% specificity.

CONCLUSION

We believe that pityriasis alba and atopic dermatitis should be considered different clinical forms within the spectrum of atopic disease, in which sun radiation plays an important role by modulating the progression of the disease.     

Observational descriptive study of cutaneous manifestations in patients from Mato Grosso with viral chronic hepatitis

BACKGROUND

Extrahepatic manifestations are seen in association with chronic infection by hepatitis B or C virus including cutaneous disorders. The frequency of these findings seems to vary among different places and reports. There is a lack of information about this issue in Brazil.

OBJECTIVES

To estimate the prevalence of cutaneous findings affecting HBV or HCV carriers from a reference outpatient unit in Mato Grosso.

METHODS

A cross-sectional observational study.

RESULTS

108 patients were studied. 88.9% presented some cutaneous findings but must of them were nonrelated to chronic viral infection. Four patients had cutaneous or autoimmune syndromes that may be HBV or HCV related.

CONCLUSION

In our study we found no statistical association between viral hepatitis and skin diseases.     

Evaluating the effectiveness of the customized Unna boot when treating patients with venous ulcers

BACKGROUND

Lower limb ulcers are a serious medical and socioeconomic problem throughout the world. One type of chronic wound of the lower extremities is the venous ulcer. Therapeutic methods for treating venous ulcer include the use of the Unna boot.

OBJECTIVES

To evaluate the effectiveness of the customized Unna boot in the treatment of venous ulcers and to monitor the subsequent development and healing of the lesions.

METHODS

Prospective exploratory and quantitative longitudinal study, conducted at the "Outpatients Department (Wound Care) of the Grupo da Fraternidade Espírita Irmão Alexandre" in the city of Pouso Alegre (MG), Brazil, in 2008. The sample consisted of 32 patients with venous ulcers who underwent treatment with the Unna boot and 11 patients (control group), who used a simple bandage application. The patients''lesions were monitored over a three month period.

RESULTS

The average age of the predominently female (65.1%) patients was 61.88. From observing the differences in healing times at the three evaluation stages, it was clear that after the initial evaluation the wound area decreased in Groups 1 and 2 (p>0.05).

CONCLUSION

The use of the customized Unna boot contributes to quicker healing. However, over a period of three months the simple bandage applications were seen to be just as effective as the Unna boot method.     

Notch Intracellular Domain Expression in Various Skin Fibroproliferative Diseases

Background

The effects of the Notch signaling pathway in fibroproliferative skin diseases have not been fully elucidated.

Objective

The aim of this study was to investigate the expression of activated Notch signaling molecules in various skin fibroproliferative diseases.

Methods

Immunohistochemical analysis of Notch intracellular domain (NICD) expression in keloid, hypertrophic scar, morphea, dermatofibroma, and normal control skin specimens was performed, and the clinical characteristics of patients with various skin fibroproliferative diseases were analyzed.

Results

NICD was highly expressed in fibroblasts of keloids and moderately to highly expressed in hypertrophic scars and dermatofibromas, whereas low or no expression was detected in the fibroblasts of normal skin specimens and morpheas. NICD was constitutively expressed in keratinocytes, endothelial cells, and immune cells in normal skin specimens.

Conclusion

NICD was significantly expressed in human fibroproliferative skin disorders, especially keloids, suggesting that an activated Notch signaling pathway is involved in the pathogenesis of skin fibrosis.