Deletion of the α2A/α2C‐adrenoceptors accelerates cutaneous wound healing in mice

The α2‐adrenoceptors regulate the sympathetic nervous system, controlling presynaptic catecholamine release. However, the role of the α2‐adrenoceptors in cutaneous wound healing is poorly understood. Mice lacking both the α2A/α2C‐adrenoceptors were used to evaluate the participation of the α2‐adrenoceptor during cutaneous wound healing. A full‐thickness excisional lesion was performed on the dorsal skin of the α2A/α2C‐adrenoceptor knockout and wild‐type mice. Seven or fourteen days later, the animals were euthanized and the lesions were formalin‐fixed and paraffin‐embedded or frozen. Murine skin fibroblasts were also isolated from α2A/α2C‐adrenoceptor knockout and wild‐type mice, and fibroblast activity was evaluated. The in vivo study demonstrated that α2A/α2C‐adrenoceptor depletion accelerated wound contraction and re‐epithelialization. A reduction in the number of neutrophils and macrophages was observed in the α2A/α2C‐adrenoceptor knockout mice compared with wild‐type mice. In addition, α2A/α2C‐adrenoceptor depletion enhanced the levels of nitrite and hydroxyproline, and the protein expression of transforming growth factor‐β and vascular endothelial growth factor. Furthermore, α2A/α2C‐adrenoceptor depletion accelerated blood vessel formation and myofibroblast differentiation. The in vitro study demonstrated that skin fibroblasts isolated from α2A/α2C‐adrenoceptor knockout mice exhibited enhanced cell migration, α‐smooth muscle actin _protein expression and collagen deposition compared with wild‐type skin fibroblasts. In conclusion, α2A/α2C‐adrenoceptor deletion accelerates cutaneous wound healing in mice.     

Dermal Transforming Growth Factor-β Responsiveness Mediates Wound Contraction and Epithelial Closure

Stromal-epithelial interactions are important during wound healing. Transforming growth factor-β (TGF-β) signaling at the wound site has been implicated in re-epithelization, inflammatory infiltration, wound contraction, and extracellular matrix deposition and remodeling. Ultimately, TGF-β is central to dermal scarring. Because scarless embryonic wounds are associated with the lack of dermal TGF-β signaling, we studied the role of TGF-β signaling specifically in dermal fibroblasts through the development of a novel, inducible, conditional, and fibroblastic TGF-β type II receptor knockout (Tgfbr2^dermalKO) mouse model. Full thickness excisional wounds were studied in control and Tgfbr2^dermalKO back skin. The Tgfbr2^dermalKO wounds had accelerated re-epithelization and closure compared with controls, resurfacing within 4 days of healing. The loss of TGF-β signaling in the dermis resulted in reduced collagen deposition and remodeling associated with a reduced extent of wound contraction and elevated macrophage infiltration. Tgfbr2^dermalKO and control skin had similar numbers of myofibroblastic cells, suggesting that myofibroblastic differentiation was not responsible for reduced wound contraction. However, several mediators of cell-matrix interaction were reduced in the Tgfbr2^dermalKO fibroblasts, including α1, α2, and β1 integrins, and collagen gel contraction was diminished. There were associated deficiencies in actin cytoskeletal organization of vasodilator-stimulated phosphoprotein-containing lamellipodia. This study indicated that paracrine and autocrine TGF-β dermal signaling mechanisms mediate macrophage recruitment, re-epithelization, and wound contraction.Skin wound healing in adult humans after full-thickness injury results in scar tissue, which is prone to contracture, loss in elasticity, and altered tensile strength. The characteristics of a healed wound, unlike the original dermal tissue, include an extracellular matrix without the normal basket weave organization of collagen fibers that can be further exaggerated as a hypertrophic scar. In scars, there is a loss of normal tissue architecture and function due, at least in part, to altered cell-matrix interaction and the assembly of the actin cytoskeleton that occurs during wound repair.^1,2 The actin cytoskeleton is important in processes that are fundamental to wound healing: migration; contraction; adhesion; and proliferation.³ The vasodilator-stimulated phosphoprotein (VASP) is associated with filamentous actin formation and likely plays a role in cell adhesion and motility.⁴ VASP may also be involved in the intracellular signaling pathways that regulate integrin-extracellular matrix interactions.The importance of stromal-epithelial interactions in development and wound healing is well established. These interactions likely involve autocrine and paracrine action of multiple growth factors, including members of the transforming growth factor-β (TGF-β) family. TGF-β1, β2, and β3 isoforms signal by binding to the TGF-β receptors type I and type I at the cell surface. The ligand-receptor complex formed stimulates multiple parallel signal pathways in the cytoplasm. TGF-β is a growth inhibitor for many cell types and stimulates extracellular matrix expression in mesenchymal cells. In the injured skin, macrophages, endothelium, fibroblasts, and epithelia are all sources of elevated TGF-β expression. TGF-β signaling at the wound site is thought to be important for extracellular matrix deposition and remodeling. The application of TGF-β1 to wounds has been shown to accelerate wound healing.⁵ In apparent contradiction, a genetic knockout of the TGF-β effector, Smad3, in mice exhibited accelerated skin and lens wound healing.^6–8 However, the epithelial and stromal compartments can respond differentially to TGF-β signaling, and the specific mechanism of TGF-β action on any individual cell type within tissues is still not understood. Wounding of embryonic skin, in contrast to that in adults, is not associated with remodeling deficiencies associated with scarring. Interestingly, embryonic dermal fibroblasts lack expression of TGF-β receptor type II.^9,10In the present study, we specifically address the role of TGF-β signaling in dermal fibroblasts by the development of a novel dermal fibroblast-inducible, conditional, TGF-β type II receptor knockout (Tgfbr2^dermalKO) mouse model. The generation of Tgfbr2^dermalKO mice provided an opportunity to test the role of TGF-β in stromal cells in wound healing. The full-thickness skin excision wounds in Tgfbr2^dermalKO mice had accelerated re-epithelization compared with control, impaired collagen organization associated with a lack of wound contraction, and reduced macrophage recruitment. The studies indicate that paracrine and autocrine TGF-β dermal responsiveness mechanisms impact skin wound healing.     

Morphofunctional Evaluation of the Effect of Transplantation of Allogeneic Fibroblasts on Healing of Burn Wounds in Albino Outbred Mice and Mutant Hr hr /Hr hr Mice

Morphofunctional evaluation of the effect of transplantation of allogeneic fibroblasts on skin regeneration after burn injury in albino outbred mice and mutant Hr ^hr /Hr ^hr (nude) mice was carried out using the radioautographic method. The pronounced stimulating effect of allogeneic fibroblasts on healing of burn wound observed in mice of both groups can be attributed to the action of bioactive substances released from destructed allogeneic fibroblasts and stimulating proliferation of stem/progenitor cells in the wound (epithelial cells in skin appendages and dermal cells in microvessel walls) at the early stages of healing.     

17beta-estradiol regulates the secretion of TGF-beta by cultured human dermal fibroblasts

Estrogen plays an important role in skin homeostasis, as demonstrated by the changes seen in the skin of post-menopausal women, changes reversed by HRT. Estrogen also has a role in wound healing, since estrogen deficiency as occurs post-menopausally and in ovariectomised animals, is associated with a reduced rate of wound healing. Estrogen appears to modulate all phases of wound healing with effects on inflammatory cells, epithelialization, angiogenesis, extracellular matrix deposition and tissue remodelling. This study was designed to investigate the effects of 17beta-estradiol on cultured human dermal fibroblasts using an in vitro wound-healing assay. The end points investigated were cell migration, proliferation, total collagen secretion and active TGF-beta1 secretion. 17beta-estradiol significantly increased the migration and proliferation of cultured dermal fibroblasts following mechanical wounding, although the secretion of total soluble collagen was not altered. An increase in TGF-beta1 was demonstrated by unwounded confluent dermal fibroblast monolayers in response to 17beta-estradiol, but paradoxically, a decrease in the secretion of TGF-beta1 was demonstrated in the mechanically wounded dermal fibroblasts. These results identify human dermal fibroblasts as estrogen target cells and provide further evidence for a role by which estrogen regulates this particular cell type as part of the wound-healing process. However, the paradoxical nature of the effect of estrogen on TGF-beta1 secretion following mechanical wounding suggests that the cellular mechanism of action is complex. A greater understanding of the cell-specific action of estrogen may help to develop therapies that will improve cutaneous wound healing in the future.     

Altered dermal fibroblast behavior in a collagen V haploinsufficient murine model of classic Ehlers–Danlos syndrome

Mutations in collagen V are associated with classic Ehlers–Danlos syndrome (EDS). A significant percentage of these mutations result in haploinsufficiency for collagen V. The purpose of this work was to determine if changes in collagen V expression are associated with altered dermal fibroblast behavior contributing to the poor wound healing response. A haploinsufficient Col5a1^+/? mouse model of EDS was utilized. In vivo wound healing studies demonstrated that mutant mice healed significantly slower than Col5a1^+/+ mice. The basis for this difference was examined in vitro using dermal fibroblast strains isolated from Col5a1^+/? and Col5a1^+/+ mice. Fibroblast proliferation was determined for each strain by counting cells at different time points after seeding as well as using the proliferation marker Ki-67. Fibroblast attachment to collagens I and III and fibronectin also was analyzed. In addition, in vitro scratch wounds were used to analyze fibroblast wound closure. Significantly decreased fibroblast proliferation was observed in Col5a1^+/? compared to Col5a1^+/+ fibroblasts. Our data indicate that the decreased fibroblast number was not due to apoptosis. Wildtype Col5a1^+/+ fibroblasts attached significantly better to components of the wound matrix (collagens I and III and fibronectin) than Col5a1^+/? fibroblasts. A significant difference in in vitro scratch wound closure rates also was observed. Col5a1^+/+ fibroblasts closed wounds in 22 h, while Col5a1^+/? fibroblasts demonstrated ~80% closure. There were significant differences in closure at all time points analyzed. Our data suggest that decreased fibroblast proliferation, extracellular matrix attachment, and migration contribute to the decreased wound healing response in classic EDS.     

Physical plasma therapy accelerates wound re-epithelialisation and enhances extracellular matrix formation in cutaneous skin grafts

Skin grafting is a surgical method of cutaneous reconstruction, which provides volumetric replacement in wounds unable to heal by primary intention. Clinically, full-thickness skin grafts (FTSGs) are placed in aesthetically sensitive and mechanically demanding areas such as the hands, face, and neck. Complete or partial graft failure is the primary complication associated with this surgical procedure. Strategies aimed at improving the rate of skin graft integration will reduce the incidence of graft failure. Cold atmospheric plasma (CAP) is an emerging technology offering innovative clinical applications. The aim of this study was to test the therapeutic potential of CAP to improve wound healing and skin graft integration into the recipient site. In vitro models that mimic wound healing were used to investigate the ability of CAP to enhance cellular migration, a key factor in cutaneous tissue repair. We demonstrated that CAP enhanced the migration of epidermal keratinocytes and dermal fibroblasts. This increased cellular migration was possibly induced by the low dose of reactive oxygen and nitrogen species produced by CAP. Using a mouse model of burn wound reconstructed with a full-thickness skin graft, we showed that wounds treated with CAP healed faster than did control wounds. Immunohistochemical wound analysis showed that CAP treatment enhanced the expression of the dermal–epidermal junction components, which are vital for successful skin graft integration. CAP treatment was characterised by increased levels of Tgfbr1 mRNA and collagen I protein in vivo, suggesting enhanced wound maturity and extracellular matrix deposition. Mechanistically, we show that CAP induced the activation of the canonical SMAD-dependent TGF- β 1 pathway in primary human dermal fibroblasts, which may explain the increased collagen I synthesis in vitro. These studies revealed that CAP improved wound repair and skin graft integration via mechanisms involving extracellular matrix formation. CAP offers a novel approach for treating cutaneous wounds and skin grafts. © 2020 The Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Cells and tissue interactions with glycated collagen and their relevance to delayed diabetic wound healing

Dermal accumulation of advanced glycation end products (AGEs) has increasingly been implicated as the underlying cause of delayed diabetic wound healing. Devising an in vitro model to adequately mimic glycated tissues will facilitate investigation into the mechanism of glycation in conjunction with exploration of new approaches or improvement of current therapies for treating diabetic chronic wounds. Collagen matrices were artificially glycated and the presence of AGEs was demonstrated by immunostaining. Both the mechanical properties of the collagen matrices and their interactions with fibroblasts (morphology, attachment, proliferation, and migration) were altered after glycation, moreover, there was evidence of impairment on extracellular matrix (ECM) remodeling as well as inhibition of cell-induced material contraction. The actin cytoskeletons of the fibroblasts residing in the glycated collagen matrices were reorganized. In vivo mice full-thickness dermal wound models implanted with glycated collagen matrices showed delayed wound healing response. Thus, the glycated collagen matrix is an adequate in vitro model to mimic glycated tissues and could serve as a facile experimental tool to investigate the mechanism of glycation in conjunction with exploration of new approaches or improvement of current therapies for treating diabetic wounds.     

Role of collagen cross-linking on equine wound contraction and healing

The present study was carried out to evaluate the effect of collagen cross-linking inhibition on equine wound contraction and healing. In five male adult donkeys, two full-thickness skin wounds (20 × 20 mm in diameter) were created on the lateral aspect of forelimbs, at the mid-point between the carpal and fetlock joints under general anesthesia. Two other wounds were created on the neck of each donkey symmetrically. Left-side wounds (test group) and right-side wounds (control group) were treated topically with beta-aminopropionitrile fumarate, 5 mg/ml, added to methyl cellulose gel and only methyl cellulose gel, respectively. Treatment of wounds were started at 24 h after wounding and continued every other day for ten successive days. The wounds were evaluated over a 3-week period. On days 0, 1, 3, 5, 7, 9, 12, 15, 18, and 21, digital photographs were taken of all wounds after careful shaving to visualize the wound margin. Rulers were held vertically and horizontally close to the wound as a reference. Epithelialization and granulation tissue formation were measured for each wound using Scion Image software. Percentage of the wound contraction, epithelialization, and healing were calculated for each wounds. At the end of the study, biopsy was taken from the center of each wound for hydroxyproline measurement and the same corner of each wound for histopathological examination. Macroscopic evaluation revealed significant differences in wound contraction and healing process between test and control groups in wounds located in neck (P < 0.05), but there was no significant difference in percent of epithelialization at the same area (P > 0.05). Significant differences were observed in the percent of wound contraction and healing between the test and control groups in wounds located in the forelimb (P < 0.05), but no significant difference was observed in percent of epithelialization at this area (P > 0.05). There were no significant differences between median of hydroxyproline levels of left and right wounds in forelimb and neck (P > 0.05). Histopathological examination revealed no significant differences between median of epithelialization, inflammatory infiltration, presence of dermal granulation tissue, fibroblast proliferation, arrangement of fibroblasts, collagen deposition, and collagen bundle formation scores in the specimens prepared from left and right wounds in forelimb and neck (P > 0.05). Our data demonstrated that collagen cross-linking could play a key role in equine wound contraction and healing at the limb and neck area.     

Contribution of bone marrow-derived cells to skin: collagen deposition and wound repair

The bone marrow provides inflammatory cells and endothelial progenitor cells to healing cutaneous wounds. To further explore the bone marrow contribution to skin and healing wounds, we used a chimeric mouse model in which the bone marrow from enhanced green fluorescent protein (EGFP) transgenic mice is transplanted into normal C57BL mice. We found that normal skin is a target organ for bone marrow-derived cells from both the hematopoietic and the mesenchymal stem cell pool. We present evidence that the bone marrow contribution to normal skin and the healing cutaneous wound is substantially greater than the previously recognized CD45+ subpopulation, where 15%-20% of the spindle-shaped dermal fibroblasts were bone marrow-derived (EGFP+). Furthermore, the bone marrow-derived cells were able to contract a collagen matrix and transcribe both collagen types I and III, whereas the skin-resident cells transcribed only collagen type I. Whereas endothelial progenitor cells were found early during the wound repair process, bone marrow-derived endothelial cells were not seen after epithelialization was complete. Our data show that wound healing involves local cutaneous cells for reconstituting the epidermis but distant bone marrow-derived cells and the adjacent uninjured dermal mesenchymal cells for reconstituting the dermal fibroblast population.     

Tissue regeneration using macrophage migration inhibitory factor-impregnated gelatin microbeads in cutaneous wounds

Migration inhibitory factor (MIF) responds to tissue damage and regulates inflammatory and immunological processes. To elucidate the function of MIF in cutaneous wound healing, we analyzed MIF knockout (KO) mice. After the excision of wounds from the dorsal skin of MIF KO and wild-type (WT) mice, healing was significantly delayed in MIF KO mice compared to WT mice. Lipopolysaccharide treatment significantly increased [(3)H]thymidine uptake in WT mouse fibroblasts compared to MIF KO mouse fibroblasts. Furthermore, there was a significant reduction in fibroblast and keratinocyte migration observed in MIF KO mice after 1-oleoyl-2-lysophosphatidic acid treatment. We subsequently examined whether MIF-impregnated gelatin slow-release microbeads could accelerate skin wound healing. Injection of more than 1.5 microg/500 microl of MIF-impregnated gelatin microbeads around a wound edge accelerated wound healing compared to a single MIF injection without the use of microbeads. MIF-impregnated gelatin microbeads also accelerated skin wound healing in C57BL/6 mice and diabetic db/db mice. Furthermore, incorporating MIF-impregnated gelatin microbeads into an artificial dermis implanted into MIF KO mice accelerated procollagen production and capillary formation. These findings suggest that MIF is crucial in accelerating cutaneous wound healing and that MIF-impregnated gelatin microbeads represent a promising treatment to facilitate skin wound healing.     

Higher numbers of autologous fibroblasts in an artificial dermal substitute improve tissue regeneration and modulate scar tissue formation

Cultured skin substitutes are increasingly important for the treatment of burns and chronic wounds. The role of fibroblast numbers present in a living-skin equivalent is at present unknown. The quality of dermal tissue regeneration was therefore investigated in relation to the number of autologous fibroblasts seeded in dermal substitutes, transplanted instantaneously or precultured for 10 days in the substitute. A full-thickness porcine wound model was used to compare acellular dermal substitutes (ADS) with dermal substitutes seeded with fibroblasts at two densities, 1x10(5) (0-DS10) and 5x10(5) cells/cm(2) (0-DS50), and with dermal substitutes seeded 10 days before operation at the same densities (10-DS10 and 10-DS50) (n=7 for each group, five pigs). After transplantation of the dermal substitutes, split-skin mesh grafts were applied on top. Wound healing was evaluated blind for 6 weeks. Cosmetic appearance was evaluated and wound contraction was measured by planimetry. The wound biopsies taken after 3 weeks were stained for myofibroblasts (alpha-smooth muscle actin), and after 6 weeks for scar tissue formation (collagen bundles organized in parallel and the absence of elastin staining). Collagen maturation was investigated with polarized light. For wound cosmetic parameters, the 10-DS50 and 0-DS50 treatments scored significantly better than the ADS treatment, as did the 10-DS50 treatment for wound contraction (p<0.05, paired t-test). Three weeks after wounding, the area with myofibroblasts in the granulation tissue, determined by image analysis, was significantly smaller for 0-DS50, 10-DS10, and 10-DS50 than for the ADS treatment (p<0.04, paired t-test). After 6 weeks, the wounds treated with 0-DS50, 0-DS10, and 10-DS50 had significantly less scar tissue and significantly more mature collagen bundles in the regenerated dermis. This improvement of wound healing was correlated with the higher numbers of fibroblasts present in the dermal substitute at the moment of transplantation. In conclusion, dermal regeneration of experimental full-skin defects was significantly improved by treatment with dermal substitutes containing high numbers of (precultured) autologous fibroblasts.     

Morphological alterations of the reticular thalamic nucleus in Engrailed-2 knockout mice

The reticular thalamic nucleus (Rt) is a sheet of neurons that surrounds the dorsal thalamus laterally, along its dorso-ventral and rostro-caudal axes. It consists of inhibitory neurons releasing gamma-aminobutyric acid (GABA). This nucleus participates in the circuitry between the thalamus and the cerebral cortex, and its impairment is associated with neuro-psychiatric disorders. In this study, we investigated the Rt anatomy of Engrailed-2 knockout mice (En2^−/−), a mouse model of autism spectrum disorder (ASD), using parvalbumin as an immunohistochemical marker. We compared 4- and 6-week-old wild type (WT) and En2^−/− mice using various morphometric parameters: cell area, shape factor, circularity and cell density. Significant differences were present in 6-week-old male mice with different genetic background (WT vs. En2^−/−): the Rt neurons of En2^−/− mice showed a bigger cell area, shape factor and circularity when compared with WT. Age (4 weeks vs. 6 weeks) influenced the shape factor of WT females, the circularity and cell density of En2^−/− males, and the shape factor and circularity of En2^−/− females. Gender affected cell density in 4-week-old WT mice, shape factor and cellularity of 6-week-old WT mice, and cell area, shape factor and cell density of En2^−/− at 6 weeks. Intrasubject (left–right) asymmetry of Rt was never observed. These results show for the first time that sex- and age-related changes occur in the Rt GABAergic neurons of the En2^−/− ASD mouse model.     

Dextran derivatives modulate collagen matrix organization in dermal equivalent

Dextran derivatives can protect heparin binding growth factor implied in wound healing, such as transforming growth factor-β1 (TGF-β1) and fibroblast growth factor-2 (FGF-2). The first aim of this study was to investigate the effect of these compounds on human dermal fibroblasts in culture with or without TGF-β1. Several dextran derivatives obtained by substitution of methylcarboxylate (MC), benzylamide (B) and sulphate (Su) groups were used to determine the effects of each compound on fibroblast growth in vitro. The data indicate that sulphate groups are essential to act on the fibroblast proliferation. The dextran derivative LS21 DMCBSu has been chosen to investigate its effect on dermal wound healing process. Fibroblasts cultured in collagenous matrices named dermal equivalent were treated with the bioactive polymer alone or associated to TGF-β1 or FGF-2. Cross-sections of dermal equivalent observed by histology or immunohistochemistry, demonstrated that the bioactive polymer accelerates the collagen matrices organization and stimulates the human type-III collagen expression. This bioactive polymer induces apoptosis of myofibroblast, property which may be beneficial in treatment of hypertrophic scar. Culture media analyzed by zymography and Western blot showed that this polymer significantly increases the secretion of zymogen and active form of matrix metalloproteinase-2 (MMP-2), involved in granulation tissue formation. These data suggest that this bioactive polymer has properties which may be beneficial in the treatment of wound healing.     

Exploration of the wound healing effect of topical administration of nicotine in combination with collagen scaffold in a rabbit model

Nicotine has been reported to prolong the wound healing; however, we showed that the topical application of 10^?4 M nicotine promoted murine wound healing. The objective of this study was to explore the wound healing effects of nicotine in combination with collagen scaffold using skin defects in rabbit. Three full-thickness skin defects 8 mm in diameter were made on the rabbit auricle. Artificial dermis was applied to the defects, and 10 μl of nicotine solution (10^?5, 10^?4, and10^?3 M), bFGF solution (0.5 μg/10 μl), and both bFGF and 10^?4 M nicotine solutions were injected into the artificial dermis once daily for 7 days. Rabbits were sacrificed on day 10, 15, or 20, and the wound healing process was evaluated. bFGF was superior in the formation of the dermis-like tissue and capillaries. In nicotine groups, the epithelial length and the dermis-like tissue formations in the 10^?4 M group were superior, in contrast, those were inhibited in the 10^?3 M group. The synergistic effect of bFGF and 10^?4 M nicotine was not confirmed. This study suggests that the topical application of 10^?4 M nicotine promoted wound healing in rabbit, but the effect was not apparent compared with murine models.     

Overexpression of Lysyl Oxidase to Increase Matrix Crosslinking and Improve Tissue Strength in Dermal Wound Healing

In this study, we aimed to increase crosslinking in collagen and elastin in the extracellular matrix through overexpression of lysyl oxidase (LO) in order to improve mechanical strength in dermal wounds during healing. We had used a gene activated matrix (GAM) approach to locally deliver plasmid DNA (pDNA) complexed with polyethylenimine (PEI) in collagen gels at the wound site for localized and sustained transfection of cells involved in the healing process. We first demonstrated in vitro that PEI-pDNA complexes in collagen gels could be taken up and expressed by cultured fibroblasts for at least 20 days. In vitro studies showed that fibroblast-seeded GAMs with the LO transgene exhibited over a 3-fold increase in mechanical strength as compared with a green fluorescent protein (GFP)-transgene control. Addition of an inhibitor of LO abolished this increase. We applied this system in a rat dermal wound healing model and showed that treatment with LO-producing GAMs led to significantly enhanced mechanical strength of the wound site.     

TGF-β1-treated ADSCs-CM promotes expression of type I collagen and MMP-1, migration of human skin fibroblasts, and wound healing in vitro and in vivo

Conditioned medium from adipose-derived stem cells (ADSCs) stimulates both collagen synthesis and migration of dermal fibroblasts. However, it is still unknown whether conditioned media from tumor growth factor (TGF)-β1-treated ADSCs (TGF-β1-treated ADSCs-CM) induces increased expression of type I collagen, matrix metalloproteinase-1 (MMP-1), and migration as well as cell cycle regulatory proteins in fibroblasts, compared to non-treated ADSCs-CM. Our data showed that TGF-β1-treated ADSCs-CM promoted effectively the proliferation and migration of human skin fibroblasts, compared to non-treated ADSCs-CM. In addition the expression of MMP-1 were markedly increased by treatment of TGF-β1-treated ADSCs-CM in fibroblasts, compared to non-treated ADSCs-CM. Expression of type I collagen protein were slightly increased by treatment of TGF-β1-treated ADSCs-CM in fibroblasts. The expression of cell cycle regulators of G1/S phase transition were not markedly altered by treatment of TGF-β1-treated ADSCs-CM. Finally, artificial wounds were made using a 4-mm punch biopsy in hairless mice and TGF-β1-treated ADSCs-CM were injected into the wound area. The injection of TGF-β1-treated ADSCs-CM promoted the wound healing process in hairless mice. Taken together, our data indicated that TGF-β1-treated ADSCs-CM induced up-regulation of type I collagen and MMP-1, promoted the migration of skin fibroblasts, and thereby promoted the wound healing process in vivo. Our data indicate that TGF-β1-treated ADSCs-CM will be a component for a wound healing accelerating agent.     

Effects of platelet-rich plasma (PRP) on cutaneous regeneration and wound healing in dogs treated with dexamethasone

To evaluate the effects of platelet-rich plasma (PRP) on cutaneous regeneration and wound healing in dogs treated with dexamethasone, the present study was undertaken. Under general anesthesia, six full-thickness skin wounds were created on the back of five male adult dogs symmetrically. Left side wounds were left without any treatment, and right side wounds were treated topically with PRP jelly. Six days before creating the wounds, dogs received dexamethasone, 0.5 mg/kg IM, and every other day up to day 8 after wounding. For macroscopic evaluation, digital photographs were taken from wounds. In days 10, 17, and 24 after wounding, skin biopsies were taken from the center and corner of each wounds for hydroxyproline measurement and histopathological evaluation. No significant difference was seen in the percentage of wound contraction, epithelialization, and healing between test and control groups during the study (P > 0.05). There were no significant differences between median of hydroxyproline levels between left and right wounds in dogs treated with dexamethasone (P > 0.0.5). There were no significant differences between median of epithelialization, inflammatory cell infiltration, presence of dermal granulation tissue, fibroblast proliferation, arrangement of fibroblasts, collagen deposition, and collagen bundle formation scores, in the specimens of left and right wounds (P > 0.05). The results of the present study demonstrated that PRP did not have significant effects to promote cutaneous regeneration and wound healing in dogs treated with dexamethasone at least 16 days after last injection.     

The effect of gelatin-chondroitin sulfate-hyaluronic acid skin substitute on wound healing in SCID mice

Tissue-engineered skin substitutes provided a feasibility to overcome the shortage of skin autograft by culturing keratinocytes and dermal fibroblasts in vitro. In this study, we applied bi-layer gelatin-chondrointin-6-sulfate-hyaluronic acid (gelatin-C6S-HA) biomatrices onto the severe combined immunodeficiency (SCID) mice to evaluate its effect on promoting wound healing. Human foreskin keratinocytes and dermal fibroblasts were cultured with reconstructed skin equivalent (rSE) for 7 days. The rSE was then grafted to the dorsum of SCID mice to evaluate its biocompatibility by histologic and immunohistochemistry analysis. The results showed that human epidermis were well-developed with the expression of differentiated markers and basement membrane-specific proteins at 4 weeks. After implantation, the percentages of skin graft take were satisfactory, while cell-seeded group was better than non-cell-seeded one. The basement membrane proteins including laminin, type IV collagen, type VII collagen, integrin alpha6, and integrin beta4 were all detected at the dermal-epidermal junction, which showed a continuous structure in the 4 weeks after grafting. This bi-layer gelatin-C6S-HA skin substitute not only has positive effect on promoting wound healing, but also has high rate of graft take. This rSE would have the potential to be applied on the extensively and deeply burned patients who suffer from severe skin defect in the near future.     

Glial cells are affected more than interneurons by the loss of Engrailed 2 gene in the mouse cerebellum

Glial cells play a pivotal role in the inflammatory processes, which are common features of several neurodevelopmental and neurodegenerative disorders. Their major role in modulating neuroinflammation underscores their significance in these conditions. Engrailed-2 knockout mice (En2^−/−) are considered a valuable model for autism spectrum disorder (ASD) due to their distinctive neuroanatomical and behavioral traits. Given the higher prevalence of ASD in males, our objective was to investigate glial and interneuron alterations in the cerebellum of En2^−/− mice compared with wild-type (WT) mice in both sexes. We employed immunohistochemical analysis to assess cell density for all cell types studied and analyzed the area (A) and shape factor (SF) of microglia cell bodies. Our findings revealed the following: (a) In WT mice, the density of microglia and astrocytes was higher in females than in males, while interneuron density was lower in females. Notably, in En2-mutant mice, these differences between males and females were not present. (b) In both male and female En2^−/− mice, astrocyte density exceeded that in WT mice, with microglia density being greater only in females. (c) In WT females, microglia cell bodies exhibited a larger area and a lower shape factor compared to WT males. Remarkably, the En2 mutation did not appear to influence these sex-related differences. (d) In both male and female En2^−/− mice, we observed a consistent pattern: microglia cell bodies displayed a larger area and a smaller shape factor. Given the ongoing debate surrounding the roles of glia and sex-related factors in ASD, our observations provide valuable insights into understanding how an ASD-associated gene En2 affects specific cell types in the cerebellum.