Control of cell migration in two and three dimensions using substrate morphology

We have shown that en masse cell migration of fibroblasts on the planar surface results in a radial outward trajectory, and a spatially dependent velocity distribution that decreases exponentially in time towards the single cell value. If the cells are plated on the surface of aligned electropsun fibers above 1 μm in diameter, they become polarized along the fiber, expressing integrin receptors which follow closely the contours of the fibers. The velocity of the cells on the fibrous scaffold is lower than that on the planar surface, and does not depend on the degree of orientation. Cells on fiber smaller than 1 μm migrate more slowly than on the planar surface, since they appear to have a large concentration of receptors. True three-dimensional migration can be observed when plating the droplet on a scaffold comprises of at least three layers. The cells still continue to migrate on the fibers surfaces, as they diffuse into the lower layers of the fibrous scaffold.     

Effects of human amniotic membrane grafts combined with marrow mesenchymal stem cells on healing of full-thickness skin defects in rabbits

We have investigated the wound-healing effects of mesenchymal stem cells (MSCs) in combination with human amniotic membrane (HAM) when grafted into full-thickness skin defects of rabbits. Five defects in each of four groups were respectively treated with HAM loaded with autologous MSCs (group A), HAM loaded with allologous MSCs (group B), HAM with injected autologous MSCs (group C), and HAM with injected allologous MSCs (group D). The size of the wounds was calculated for each group at 7, 12, and 15 days after grafting. The wounds were subsequently harvested at 25 days after grafting. Sections stained with hematoxylin and eosin were used to determine the quality of wound healing, as based on the characteristics and amount of granulated tissue in the epidermal and dermal layers. Groups A and B showed the most pronounced effect on wound closure, with statistically significant improvement in wound healing being seen on post-operative days 7, 12, and 15. Although a slight trend toward improved wound healing was seen in group A compared with group B, no statistically significant difference was found at any time point between the two groups. Histological examination of healed wounds from groups A and B showed a thin epidermis with mature differentiation and collagen bundle deposition plus recovered skin appendages in the dermal layer. In contrast, groups C and D showed thickened epidermis with immature epithelial cells and increased fibroblast proliferation with only partially recovered skin appendages in the dermal layer. Thus, the graft of HAM loaded with MSCs played an effective role during the healing of skin defects in rabbits, with no significant difference being observed in wound healing between autologous and allologous MSC transplantation. This study was supported by research funds from Dong-A University.     

The plasmid encoding HSP47 enhances collagen expression and promotes skin wound healing in an alloxan-induced diabetic model

The objective of this work was initially to investigate the effects on skin wound healing process by local injection of HSP47 recombinant plasmid in an alloxan-induced diabetic rat model and assess the possibility and utility of gene therapy based on HSP47 plasmid to improve the diabetic skin wound healing. Rats were injected intraperitoneally with alloxan (120 mg/kg) to induce diabetes. The fragment containing the rat 47 kDa heat shock protein (HSP47) gene lacking its own promoter was cloned into plasmids containing a promoter and green fluorescent protein (GFP). The resulting gene constructs were first tested in vitro using 3T3 fibroblast cell line and subsequently in vivo after inducing wounds with alloxan in diabetic rats. Immunohistochemistry, quantitative fluorescent RT-PCR, and Western blotting 3-5 days after plasmid injection were performed to measure the expression changes of HSP47 and collagen I. The results demonstrate an increase of HSP47 levels in vitro in 3T3 fibroblast cells and in vivo in diabetic rat after treatment with plasmids expressing HSP47. The level of collagen I around the wound during the repair process was higher in the treated group than that in the control group, indicating that the constructs may have use in human gene therapy in cases of impaired skin wound healing in diabetes.     

Cloning and expression of rabbit CCT subunits eta and beta in healing cutaneous wounds

We have previously identified the CCT subunit eta as specifically reduced in healing fetal skin wounds by differential display, and observed that this reduction is not seen with any other CCT subunit. We now report the cloning and characterization of the cDNAs for rabbit CCT-eta and its closest evolutionary homolog, CCT-beta. Quantitative examination of CCT-eta and –beta message expression in healing fetal and adult wounds at 12 h post-injury confirms that CCT-eta mRNA is decreased in fetal wound tissues, but actually elevated in adult wound tissues. CCT-beta mRNA, in contrast, remains unchanged in both fetal and adult wound tissues. CCT-eta mRNA remains persistently elevated in healing adult wounds for 28 days following injury, whereas CCT-beta mRNA remains invariant throughout. CCT-eta protein is similarly increased, whereas CCT-beta protein remains unchanged. -smooth muscle actin (-SMA), a recognized substrate of CCT known to be important in integumentary wound healing, was also measured over the course of wound healing, and both mRNA and protein levels were elevated throughout the 28 days.     

Modulation of protein tyrosine phosphorylation in gastric mucosa during re-epithelization processes

AIM: To investigate the role of protein tyrosine phosphorylation in gastric wound formation and repair following ulceration.METHODS: Gastric lesions were induced in rats using restraint cold stress. To investigate the effect of oxidative and nitrosative cell stress on tyrosine phosphorylation during wound repair, total activity of protein tyrosine kinase (PTK), protein tyrosine phosphatase (PTP), antioxidant enzymes, nitric oxide synthase (NOS), 2’,5’-oligoadenylate synthetase, hydroxyl radical and zinc levels were assayed in parallel.RESULTS: Ulcer provocation induced an immediate decrease in tyrosine kinase (40% in plasma membranes and 56% in cytosol, P < 0.05) and phosphatase activity (threefold in plasma membranes and 3.3-fold in cytosol), followed by 2.3-2.4-fold decrease (P < 0.05) in protein phosphotyrosine content in the gastric mucosa. Ulceration induced no immediate change in superoxide dismutase (SOD) activity, 30% increase (P < 0.05) in catalase activity, 2.3-fold inhibition (P < 0.05) of glutathione peroxidase, 3.3-fold increase (P < 0.05) in hydroxyl radical content, and 2.3-fold decrease (P < 0.05) in zinc level in gastric mucosa. NOS activity was three times higher in gastric mucosa cells after cold stress. Following ulceration, PTK activity increased in plasma membranes and reached a maximum on day 4 after stress (twofold increase, P < 0.05), but remained inhibited (1.6-3-fold decrease on days 3, 4 and 5, P < 0.05) in the cytosol. Tyrosine phosphatases remained inhibited both in membranes and cytosol (1.5-2.4-fold, P < 0.05). NOS activity remained increased on days 1, 2 and 3 (3.8-, 2.6-, 2.2-fold, respectively, P < 0.05). Activity of SOD increased 1.6 times (P < 0.05) days 4 and 5 after stress. Catalase activity normalized after day 2. Glutathione peroxidase activity and zinc level decreased (3.3- and 2-fold, respectively, P < 0.05) on the last day. Activity of 2’,5’-oligoadenylate synthethase increased 2.8-fold (P < 0.05) at the beginning, and 1.6-2.3-fold (P < 0.05) during ulcer recuperation, and normalized on day 5, consistent with slowing of inflammation processes.CONCLUSION: These studies show diverse changes in total tyrosine kinase activity in gastric mucosa during the recovery process. Oxidative and nitrosative stress during lesion formation might lead to the observed reduction in tyrosine phosphorylation during ulceration.     

Effects of genistein on early-stage cutaneous wound healing

Wound healing occurs in three sequential phases: hemostasis and inflammation, proliferation, and remodeling. Inflammation, the earliest phase, is considered a critical period for wound healing because immune cells remove damaged tissues, foreign debris, and remaining dead tissue. Wound healing would be delayed without inflammation, and this phase is affected by antioxidation capacity. Therefore, we hypothesized that genistein, which has an antioxidant effect, might modulate the wound healing process by altering the inflammatory response. After three days of acclimation, mice were divided into three groups: control, 0.025% genistein, and 0.1% genistein. After two weeks of an experimental diet, skin wounds were induced. Wounded skin areas were imaged, and the healing rate calculated. To measure lipid peroxidation, antioxidant enzyme expression and activity, and pro-inflammatory cytokine expression, skin and liver tissues were harvested at 12, 24, 48, and 72 h. Genistein did not affect body weight. The rate of wound closure in mice fed genistein was significantly faster than in the control group during the early stage of wound healing, especially in first three days. Cu, Zn-SOD and Mn-SOD expression in wound skin tissue in the 0.1% genistein group was lower than in the control group. However, CAT expression did not differ among groups. We also found that genistein modulated NF-κB and TNF-α expression during the early stage of wound healing. The genistein group had significantly lower hepatic lipid peroxidation and higher SOD, CAT, and GPx activities than the control group. These results suggest that genistein supplementation reduces oxidative stress by increasing antioxidant capacity and modulating proinflammatory cytokine expression during the early stage of wound healing.     

Wound healing and blastema formation in regenerating digit tips of adult mice

Amputation of the distal region of the terminal phalanx of mice causes an initial wound healing response followed by blastema formation and the regeneration of the digit tip. Thus far, most regeneration studies have focused in embryonic or neonatal models and few studies have examined adult digit regeneration. Here we report on studies that include morphological, immunohistological, and volumetric analyses of adult digit regeneration stages. The regenerated digit is grossly similar to the original, but is not a perfect replacement. Re-differentiation of the digit tip occurs by intramembranous ossification forming a trabecular bone network that replaces the amputated cortical bone. The digit blastema is comprised of proliferating cells that express vimentin, a general mesenchymal marker, and by comparison to mature tissues, contains fewer endothelial cells indicative of reduced vascularity. The majority of blastemal cells expressing the stem cell marker SCA-1, also co-express the endothelial marker CD31, suggesting the presence of endothelial progenitor cells. Epidermal closure during wound healing is very slow and is characterized by a failure of the wound epidermis to close across amputated bone. Instead, the wound healing phase is associated with an osteoclast response that degrades the stump bone allowing the wound epidermis to undercut the distal bone resulting in a novel re-amputation response. Thus, the regeneration process initiates from a level that is proximal to the original plane of amputation.     

Pivotal role for alpha1-antichymotrypsin in skin repair

α1-Antichymotrypsin (α1-ACT) is a specific inhibitor of leukocyte-derived chymotrypsin-like proteases with largely unknown functions in tissue repair. By examining human and murine skin wounds, we showed that following mechanical injury the physiological repair response is associated with an acute phase response of α1-ACT and the mouse homologue Spi-2, respectively. In both species, attenuated α1-ACT/Spi-2 activity and gene expression at the local wound site was associated with severe wound healing defects. Topical application of recombinant α1-ACT to wounds of diabetic mice rescued the impaired healing phenotype. LC-MS analysis of α1-ACT cleavage fragments identified a novel cleavage site within the reactive center loop and showed that neutrophil elastase was the predominant protease involved in unusual α1-ACT cleavage and inactivation in nonhealing human wounds. These results reveal critical functions for locally acting α1-ACT in the acute phase response following skin injury, provide mechanistic insight into its function during the repair response, and raise novel perspectives for its potential therapeutic value in inflammation-mediated tissue damage.