Wound healing profile of topical xylocaine preparations in rodents.

Local anesthetics are commonly used for alleviating pain in many clinical conditions associated with wound. Present study is undertaken to unravel the implications of two xylocaine preparations on dermal wound repair in albino rats. Application of xylocaine either as jelly or ointment significantly reduced skin wound breaking strength and promoted re-epithelization. While bases of xylocaine jelly and ointment had no action on skin wound breaking strength but enhanced re-epithelization. Current findings, suggest that prior knowledge of the effects of substances incorporated in topical preparations on wound repair is important for their judicious use and selection of preparation in relevant clinical conditions.     

Healing of skin wounds in the African catfish Clarias gariepinus

The African catfish Clarias gariepinus was used as a model for wound healing and tissue regeneration in a scale-less fish. A temporal framework of histological and cell proliferation markers was established after wound induction in the dorsolateral cranial region, by removing the epidermal and dermal layers, including stratum adiposum (SA). Wound closure and epidermis formation was initiated within 3 h post-procedure (hpp) with migration and concomitant proliferation of epidermal cells from the wound borders. The wound was covered by this primary epidermal front 12 hpp and fusion of the opposing epidermal fronts occurred within 24 hpp. Attachment of the newly formed epidermal layer to the underlying dermis was observed 48 hpp concomitant with a second wave of cell proliferation at the wound edge. Normal epidermal thickness within the wound was achieved 72 hpp. Formation of a basement membrane occurred by 120 hpp with concomitant emergence of the SA from the wound borders. Wound healing in C. gariepinus skin involved closure of the wound and re-epithelization through cell migration with a single wave of early cell proliferation not documented in other species. Furthermore, covering of the wound by epithelium as well as the reappearance of the basement membrane and SA occurred sooner than in other fish species.     

Epidermal stem cells - role in normal, wounded and pathological psoriatic and cancer skin

In this review we focus on epidermal stem cells in the normal regeneration of the skin as well as in wounded and psoriatic skin. Furthermore, we discuss current data supporting the idea of cancer stem cells in the pathogenesis of skin carcinoma and malignant melanoma. Epidermal stem cells present in the basal layer of the interfollicular epidermis and in the bulge region of the hair follicle play a critical role for normal tissue maintenance. In wound healing, multipotent epidermal stem cells contribute to re-epithelization. It is possible that defects in growth control of either epidermal stem cells or transit amplifying cells constitute a primary pathogenetic factor in the epidermal hyperproliferation seen in psoriasis. In cutaneous malignancies mounting evidence supports a stem cell origin in skin carcinoma and malignant melanoma and a possible existence of cancer stem cells.     

Myostatin-null mice exhibit delayed skin wound healing through the blockade of transforming growth factor-β signaling by decorin

Myostatin (Mstn) is a secreted growth and differentiation factor that belongs to the transforming growth factor-β (TGF-β) superfamily. Mstn has been well characterized as a regulator of myogenesis and has been shown to play a critical role in postnatal muscle regeneration. Herein, we report for the first time that Mstn is expressed in both epidermis and dermis of murine and human skin and that Mstn-null mice exhibited delayed skin wound healing attributable to a combination of effects resulting from delayed epidermal reepithelialization and dermal contraction. In epidermis, reduced keratinocyte migration and protracted keratinocyte proliferation were observed, which subsequently led to delayed recovery of epidermal thickness and slower reepithelialization. Furthermore, primary keratinocytes derived from Mstn-null mice displayed reduced migration capacity and increased proliferation rate as assessed through in vitro migration and adhesion assays, as well as bromodeoxyuridine incorporation and Western blot analysis. Moreover, in dermis, both fibroblast-to-myofibroblast transformation and collagen deposition were concomitantly reduced, resulting in a delayed dermal wound contraction. These decreases are due to the inhibition of TGF-β signaling. In agreement, the expression of decorin, a naturally occurring TGF-β suppressor, was elevated in Mstn-null mice; moreover, topical treatment with TGF-β1 protein rescued the impaired skin wound healing observed in Mstn-null mice. These observations highlight the interplay between TGF-β and Mstn signaling pathways, specifically through Mstn regulation of decorin levels during the skin wound healing process. Thus we propose that Mstn agonists might be beneficial for skin wound repair.     

Bone marrow stromal cells, preadipocytes, and dermal fibroblasts promote epidermal regeneration in their distinctive fashions

Mesenchymal cell types, under mesenchymal-epithelial interaction, are involved in tissue regeneration. Here we show that bone marrow stromal cells (BMSCs), subcutaneous preadipocytes, and dermal fibroblasts distinctively caused keratinocytes to promote epidermal regeneration, using a skin reconstruction model by their coculture with keratinocytes. Three mesenchymal cell types promoted the survival, growth, and differentiation of keratinocytes, whereas BMSCs and preadipocytes inhibited their apoptosis. BMSCs and preadipocytes induced keratinocytes to reorganize rete ridge- and epidermal ridge-like structures, respectively. Keratinocytes with fibroblasts or BMSCs expressed the greatest amount of interleukin (IL)-1alpha protein, which is critical for mesenchymal-epithelial cross-talk in skin. Keratinocytes with or without three mesenchymal supports displayed another cross-talk molecule, c-Jun protein. Without direct mesenchymal-epithelial contact, the rete ridge- and epidermal ridge-like structures were not replicated, whereas the other phenomena noted above were. DNA microarray analysis showed that the mesenchymal-epithelial interaction affected various gene expressions of keratinocytes and mesenchymal cell types. Our results suggest that not only skin-localized fibroblasts and preadipocytes but also BMSCs accelerate epidermal regeneration in complexes and that direct contact between keratinocytes and BMSCs or preadipocytes is required for the skin-specific morphogenesis above, through mechanisms that differ from the IL-1alpha/c-Jun pathway.     

Confocal microscopic analysis of scarless repair in the fetal rat: defining the transition.

Fetal wounds pass from scarless repair to healing with scar formation during gestation. This transition depends on both the size of the wound and the gestational age of the fetus. This study defines the transition period in the fetal rat model and provides new insight into scarless collagen wound architecture by using confocal microscopy. A total of 16 pregnant Sprague-Dawley rats were operated on. Open full-thickness wounds, 2 mm in diameter, were created on fetal rats at gestational ages 14.5 days (E14; n = 10), 16.5 days (E16; n = 42), and 18.5 days (E18; n = 42) (term = 21.5 days). Wounds were harvested at 24 (n = 18 per gestational age) and 72 hours (n = 24 per gestational age). Skin at identical gestational ages to wound harvest was used for controls. The wounds were fixed and stained with hematoxylin and eosin, antibody to type I collagen, and Sirius red for confocal microscopic evaluation. No E14 rat fetuses survived to wound harvest. Wounds created on E16 fetal rats healed completely and without scarring. E16 fetal rat hair follicle formation and collagen architecture was similar to that of normal, nonwounded skin. Wounds created on E18 fetal rats demonstrated slower healing; only 50 percent were completely healed at 72 hours compared with 100 percent of the E16 fetal rat wounds at 72 hours. Furthermore, the E18 wounds healed with collagen scar formation and without hair follicle formation. Confocal microscopy demonstrated that the collagen fibers were thin and arranged in a wispy pattern in E16 fetal rat wounds and in nonwounded dermis. E18 fetal rat wounds had thickened collagen fibers with large interfiber distances. Two-millimeter excisional E16 fetal rat wounds heal without scar formation and with regeneration of normal dermal and epidermal appendage architecture. E18 fetal rat wounds heal in a pattern similar to that of adult cutaneous wounds, with scar formation and absence of epidermal appendages. Confocal microscopy more clearly defined the dermal architecture in normal skin, scarless wounds, and scars. These data further define the transition period in the fetal rat wound model, which promises to be an effective system for the study of in vivo scarless wound healing.     

Comparison of hair dermal cells and skin fibroblasts in a collagen sponge for use in wound repair

The adult hair follicle has well-defined dermal and epithelial populations that display distinct developmental properties. The follicular dermal cells, namely the dermal papilla and dermal sheath, are derived from the same mesenchymal cells as dermal fibroblasts and therefore, we believed that follicular cells could be useful sources of interfollicular keratinocytes and fibroblast for skin wound repair. In this study, we evaluated the relative effect of various mesenchymal-derived cells on wound healing following skin injury. Human dermal cells, including two different follicular dermal cells and skin fibroblasts were cultured in collagen sponges and compared with respect to wound healing. Results indicated that there was no significant difference in wound contraction and angiogenesis among the cell types. Further, dermal sheath cells exhibited relatively poor results compared with other cells in new collagen synthesis. Finally, basement membrane reformation and new collagen synthesis for the dermal papilla cell grafts was superior to those of the dermal sheath cells or fibroblasts.     

Replenishment of type VII collagen and re-epithelialization of chronically ulcerated skin after intradermal administration of allogeneic mesenchymal stromal cells in two patients with recessive dystrophic epidermolysis bullosa

In animal models it has been shown that mesenchymal stromal cells (MSC) contribute to skin regeneration and accelerate wound healing. We evaluated whether allogeneic MSC administration resulted in an improvement in the skin of two patients with recessive dystrophic epidermolysis bullosa (RDEB; OMIM 226600). Patients had absent type VII collagen immunohistofluorescence and since birth had suffered severe blistering and wounds that heal with scarring. Vehicle or 0.5 × 10⁶ MSC were infused intradermally in intact and chronic ulcerated sites. One week after intervention, in MSC-treated skin type VII collagen was detected along the basement membrane zone and the dermal–epidermal junction was continuous. Re-epithelialization of chronic ulcerated skin was observed only near MSC administration sites. In both patients the observed clinical benefit lasted for 4 months. Thus intradermal administration of allogeneic MSC associates with type VII collagen replenishment at the dermal–epidermal junction, prevents blistering and improves wound healing in unconditioned patients with RDEB.     

Objective Assessment of Endogenous Collagen In Vivo during Tissue Repair by Laser Induced Fluorescence

Collagen, a triple helical protein with the primary role of mechanical function, provides tensile strength to the skin, and plays a pivotal task in tissue repair. During tissue regeneration, collagen level increases gradually and therefore, monitoring of such changes in vivo by laser induced fluorescence was the main objective behind the present study. In order to accomplish this, 15 mm diameter excisional wounds were created on six to eight week old Swiss albino mice. The collagen deposition accelerated upon irradiation of single exposure of 2 J/cm² He-Ne laser dose immediately after wounding was recorded by laser induced autofluorescence in vivo along with un-illuminated and un-wounded controls. Autofluorescence spectra were recorded for each animal of the experimental groups on 0, 5, 10, 30, 45 and 60 days post-wounding, by exciting the granulation tissue/skin with 325 nm He-Cd laser. The variations in the average collagen intensities from the granulation tissue/skin of mice were inspected as a function of age and gender. Further, the spectral findings of the collagen synthesis in wound granulation tissue/un-wounded skin tissues were validated by Picro-Sirius red- polarized light microscopy in a blinded manner through image analysis of the respective collagen birefringence. The in vivo autofluorescence studies have shown a significant increase in collagen synthesis in laser treated animals as compared to the un-illuminated controls. Image analysis of the collagen birefringence further authenticated the ability of autofluorescence in the objective monitoring of collagen in vivo. Our results clearly demonstrate the potential of laser induced autofluorescence in the monitoring of collegen synthesis during tissue regeneration, which may have clinical implications.     

Exploring the dermal “template effect” and its structure

Scar formation is the problem for clinic surgery. Recent studies showed that the scar formation was closely related to the dermal defect. Three-dimensional (3-d) structures of dermal tissues act as a template to modulate cell functions that are essential the regeneration of skin structure and function. The dermal tissue’s integrity and continuity is a prerequisite for repair to take place. Loss of the dermal tissue integrity and continuity due to trauma leads to a lack of the template effect, which may be one important mechanism that hinders the recovery of cell function, resulting in scar formation. These studies give us two questions: what is the three-dimensional (3-d) structure of the dermal tissue? How do the tissues form? Up to now, it is well known that the molecular structure of collagen, the micro-structure of microfibril, however, the mesoscopic structure of dermal tissues is still unclear. Our recently rudimentary studies showed the problem might be resolved by phase-contrast micro-tomography with synchrotron radiation, which is likely to open new avenues for further investigations on wound regeneration and skin tissue engineering.     

Full-thickness tissue engineered skin constructed with autogenic bone marrow mesenchymal stem cells

To explore the feasibility of repairing clinical cutaneous deficiency, autogenic bone marrow mesenchymal stem cells (BMSCs) were isolated and differentiated into epidermal cells and fibroblasts in vitro supplemented with different inducing factors and biomaterials to construct functional tissueengineered skin. The results showed that after 72 h induction, BMSCs displayed morphologic changes such as typical epidermal cell arrangement, from spindle shape to round or oval; tonofibrils, melanosomes and keratohyaline granules were observed under a transmission electronic microscope. The differentiated cells expressed epidermal stem cell surface marker CK19 (59.66% ± 4.2%) and epidermal cells differentiation marker CK10. In addition, the induced epidermal cells acquired the anti-radiation capacity featured by lowered apoptosis following exposure to UVB. On the other hand, the collagen microfibrils deposition was noticed under a transmission electronic microscope after differentiating into dermis fibroblasts; RT-PCR identified collagen type I mRNA expression in differentiated cells; radioimmunoassay detected the secretion of interleukin-6 (IL-6) and interleukin-8 (IL-8) (up to 115.06 pg/mL and 0.84 ng/mL, respectively). Further in vivo implanting BMSCs with scaffold material shortened skin wound repair significantly. In one word, autogenic BMSCs have the potential to differentiate into epidermal cells and fibroblasts in vitro, and show clinical feasibility acting as epidermis-like and dermis-like seed cells in skin engineering.     

Multiple growth factor delivery for skin tissue engineering applications

Administration of exogenous growth factors (GFs) to a damaged site has been investigated for skin tissue regeneration. Among the many types of GFs and cytokines, epidermal growth factor, vascular endothelial growth factor, platelet-derived growth factor, fibroblast growth factor, and hepatocyte growth factor could be specifically used for stimulating molecules in wound healing as well as for recovery of damaged skin tissues. It is speculated that delivered GFs could stimulate various cellular functions, including proliferation, migration, deposition of extracellular matrix molecules, and remodeling of collagen synthesis. Although the physiological wound healing process is complex, engineering strategies for proper delivery of multiple therapeutic GFs could enhance the quality and quantity of regenerated skin tissues. As compared to single delivery of a GF, recent studies have proven that any combination of multiple GFs and/or therapeutic chemical factors synergistically facilitates the regeneration of damaged skin tissues. In order to maximize the stability, bioactivity, intrinsic therapeutic functionality, and efficiency of internal delivery of cargo GFs, it is essential to utilize tissueengineered biomaterials and related composites as implantable platforms. Successful fabrication and development of skin tissue engineering applications as well as subsequent surgical implantation of these platforms might provide clinical treatment for superior skin regeneration. Therefore, the present review summarizes the biological functions, related signaling mechanisms, and recent developments of tissue engineering applications for multiple GF delivery.     

Enhanced wound healing in animal models by interferon and an interferon inducer.

Healing of wounds is a significant biological process that involves the interactions of different cell types, growth factors, cytokines, and extracellular matrix molecules. Mice and rats were treated in vivo with interferon (IFN) alpha/beta or polyinosinic-polycytidylic acid (Poly I:C), a ds-RNA, a potent inducer of IFN. We observed faster and enhanced closure of wounds as compared to untreated controls on day 7 (wound area measured on Macintosh II CX using NIH image 1.30u program), increased migration of dermal fibroblasts in the wound bed, complete re-epithelialization evidenced by routine histology and scanning electron microscopic procedures, and increased collagen synthesis, which correlates to greater tensile strength. In addition, classical immunofluorescence procedures using frozen sections showed that dermal fibroblasts synthesized much more laminin following Poly I:C treatment, whereas no effect was observed on fibronectin synthesis. These results suggest that Poly I:C and IFN treatment result in a faster restoration of tissue integrity in both full skin punch biopsy and skin incision models.     

Exogenous liposomal IGF-I cDNA gene transfer leads to endogenous cellular and physiological responses in an acute wound

The purpose of the present study was to examine whether exogenous liposomal cDNA gene transfer is recognized by the cell and causes endogenous cellular and physiological responses. When administered as a protein, IGF-I is known to cause adverse side effects due to lack of cellular responses. Therefore, we used IGF-I cDNA as a vector to study cellular and physiological effects after liposomal administration to wounded skin. Sprague-Dawley rats were given a scald burn to inflict an acute wound and were divided into two groups to receive weekly subcutaneous injections of liposomes plus the Lac-Z gene (0.2 microg vehicle) or liposomes plus the IGF-I cDNA (2.2 microg) and Lac Z gene (0.22 microg). Transfection was confirmed by histochemical assays for beta-galactosidase. Planimetry, immunological assays, and histological and immunohistochemical techniques were used to determine molecular mechanisms after gene transfer, protein expression, and dermal and epidermal regeneration. IGF-I cDNA transfer increased IGF-I protein expression and caused concomitant cellular responses by increasing IGF binding protein (IGFBP)-3 and decreasing IGFBP-1. IGF-I cDNA gene transfer increased keratinocyte growth factor expression and exerted promitogenic antiapoptotic effects on basal keratinocytes, thus improving epidermal regeneration. IGF-I cDNA improved dermal regeneration by an increased collagen deposition and morphology. IGF-I cDNA increased VEGF concentrations and thus neovascularization. Exogenous-administered IGF-I cDNA is recognized by the cell and leads to similar intracellular responses as the endogenous gene. Liposomal IGF-I gene transfer further leads to improved dermal and epidermal regeneration by interacting with other growth factors.     

Atropa belladonna L. water extract: modulator of extracellular matrix formation in vitro and in vivo

Previously, we found that treatment of cutaneous wounds with Atropa belladonna L. (AB) revealed shortened process of acute inflammation as well as increased tensile strength and collagen deposition in healing skin wounds (Gál et al. 2009). To better understand AB effect on skin wound healing male Sprague-Dawley rats were submitted to one round full thickness skin wound on the back. In two experimental groups two different concentrations of AB extract were daily applied whereas the control group remained untreated. For histological evaluation samples were removed on day 21 after surgery and stained for wide spectrum cytokeratin, collagen III, fibronectin, galectin-1, and vimentin. In addition, in the in vitro study different concentration of AB extract were used to evaluate differences in HaCaT keratinocytes proliferation and differentiation by detection of Ki67 and keratin-19 expressions. Furthermore, to assess ECM formation of human dermal fibroblasts on the in vitro level fibronectin and galectin-1 were visualized. Our study showed that AB induces fibronectin and galectin-1 rich ECM formation in vitro and in vivo. In addition, the proliferation of keratinocytes was also increased. In conclusion, AB is an effective modulator of skin wound healing. Nevertheless, further research is needed to find optimal therapeutic concentration and exact underlying mechanism of action.     

Full-thickness tissue engineered skin constructed with autogenic bone marrow mesenchymal stem cells

To explore the feasibility of repairing clinical cutaneous deficiency, autogenic bone marrow mesenchymal stem cells (BMSCs) were isolated and differentiated into epidermal cells and fibroblasts in vitro supplemented with different inducing factors and biomaterials to construct functional tissueengineered skin. The results showed that after 72 h induction, BMSCs displayed morphologic changes such as typical epidermal cell arrangement, from spindle shape to round or oval; tonofibrils, melanosomes and keratohyaline granules were observed under a transmission electronic microscope. The differentiated cells expressed epidermal stem cell surface marker CK19 (59.66% ± 4.2%) and epidermal cells differentiation marker CK10. In addition, the induced epidermal cells acquired the anti-radiation capacity featured by lowered apoptosis following exposure to UVB. On the other hand, the collagen microfibrils deposition was noticed under a transmission electronic microscope after differentiating into dermis fibroblasts; RT-PCR identified collagen type I mRNA expression in differentiated cells; radioimmunoassay detected the secretion of interleukin-6 (IL-6) and interleukin-8 (IL-8) (up to 115.06 pg/mL and 0.84 ng/mL, respectively). Further in vivo implanting BMSCs with scaffold material shortened skin wound repair significantly. In one word, autogenic BMSCs have the potential to differentiate into epidermal cells and fibroblasts in vitro, and show clinical feasibility acting as epidermis-like and dermis-like seed cells in skin engineering. Supported by the Major Technology Program of Beijing Municipal Science & Technology Commission (Grant No. H060920050130) and the Major State Basic Research Development Program of China (Grant No. 2005CB522702)     

The tetrachlorodecaoxygen complex reverses the effect of cortisone on wound healing

Immunosuppression induced by the administration of glucocorticoids will prevent normal wound contraction and normal increases in tensile strength. Vitamin A, anabolic steroids, and growth hormone will, in the presence of glucocorticoids, restore mesenchymal cell proliferation, the accumulation of collagen, and the rate of increase of wound tensile strength. They will not, however, antagonize the inhibition of wound contraction. A novel inorganic agent, the tetrachlorodecaoxygen anion complex (TCDO), known to enhance the migration and activation of macrophages, was tested in a rat model of impaired wound healing using high doses of glucocorticoids. Histology, changes in wound contraction, collagen synthesis, and tensile strength were evaluated. Animals receiving cortisone in combination with TCDO displayed markedly enhanced wound healing, including restoration of tensile strength, collagen synthesis, and wound contraction. The results indicate that TCDO could be a potential agent of wound healing in immunosuppressed patients and anergic wounds.     

Systemically transplanted bone marrow stromal cells contributing to bone tissue regeneration

Bone marrow stromal cells (BMSCs) are a rich source of osteogenic progenitor cells. A fundamental question is whether systemically transplanted BMSCs participate in bone regeneration. Luciferase and GFP double-labeled BMSCs were transplanted into irradiated mice. Five weeks after transplantation, artificial bone wounds were created in the mandibles and calvaria of the recipients. Animals were sacrificed at weeks 2, 4, and 6 after surgery and the expressions of luciferase and GFP were determined using Xenogen IVIS Imaging System, immunohistochemical staining and RT-PCR. The results demonstrated that transplanted BMSCs can be detected in wound sites as early as 2 weeks and lasted the whole experimental period. Luciferase expression peaked at 2 weeks after surgery and decreased thereafter, exhibiting a similar expression pattern as that of BSP, while GFP expression was relatively stable during the experimental period. In conclusion, BMSCs can migrate to bone wound sites and participate in bone regeneration in orocraniofacial region.