Healing Wound Tissue Research Articles

Effect of rat adipose-derived mesenchymal stem cells on macrophages phenotype in skin wound

Objective To investigate the effect of local injection of rat adipose-derived mesenchymal stem cells (ADSCs)on the phenotype of macrophages in skin wound. Methods The cryopreserved primary SD rat ADSCs were resuscitated and then sub-cultured. ADSCs of the third generation were used in the experiments. Thirty six SD rats were divided into ADSCs group (n=18) and control group(n=18) by random numbers table method. The full thickness skin wounds were established on both sides of the spine. After the model establishment 0.2 ml ADSCs suspension labeled by live cell stain Chloromethylbenzamido derivatives of 1, 1'-dioctadecyl-3, 3, 3', 3'-tetramethylindo-carbocyanine perchlorate (CM-Dil) with the concentration of 5×106/ml was subcutaneously annularly injected in the skin wound of SD rats in ADSCs group. The SD rats in control group were given 0.2 ml serum-free Dulbecco modified Eagle medium (DMEM). On 3, 7, and 14 days after injury, six rats were selected from each group to measure the wound area and healing rate. The healed wound tissues were harvested to observe the morphology by HE staining. The expressions of interleukin (IL)-10 were detected by immunohistochemistry staining. The double-positive expressions of CD68 and inducible nitric oxide synthase (iNOS) (M1 type macrophages) and of CD68 and arginase-1 (Arg-1) (M2 type macrophages) were detected by immunofluorescent staining. The distribution of CM-Dil-labeled ADSCs in healed wound tissue 14 days after injury was observed by inverted fluorescence microscope. Results (1) At day 3 after injury, the wound areas in two groups were covered with crust and surrounding redness, and the wound healing rates were slightly different; at day 7 after injury, the wound area of ADSCs group was significantly smaller than that of control group, and the healing speed and rate of ADSCs group was significantly higher than that of control group (P<0.01); at day 14 after injury, the healing rate of ADSCs group was nearly 99% (P<0.01), and the healing skin tissue texture of ADSCs group was better than that of control group. (2) At day 3 after injury, there were a large number of inflammatory cells and disorganized collagen fiber in the wound areas of two groups; at day 7 after injury, the inflammatory cells infiltration reduced in ADSCs group compared with control group, and the collagen fiber arrangement in control group was in disorder; at day 14 after injury, the inflammatory cells in both groups obviously decreased, and ADSCs group had more new vessels and more orderly arrangement of collagen fiber than control group. CM Dil labeled ADSCs were seen in the healing wound tissue in ADSCs group. (3) At day 3 after injury, there was little difference in M1 type macrophage distribution in the two groups; ADSCs group had more M2 type macrophage cells than control group significantly (P<0.01); the expression of IL 10 in ADSCs group was not high, which did not differ from that of control group; at days 7 and 14 after injury, ADSCs group has fewer M1 type macrophage cells, more M2 type macrophage cells, and higher expressions of IL-10 than control group (P<0.01). Conclusion The ADSCs trasplantation can promote the change from M1 type to M2 type macrophages, facilitating wound regeneration and healing. Key words: Mesenchymal stem cells; Macrophages; Wound healing

Effects of human amniotic epithelial stem cells-derived exosomes on healing of wound with full-thickness skin defect in rats

Objective: To investigate the effects of human amniotic epithelial stem cells-derived exosomes on healing of wound with full-thickness skin defect in rats. Methods: (1) Human amniotic epithelial stem cells were isolated from the amnion tissue of 5 full-term pregnant women in Department of Obstetrics of our hospital by the method of trypsin digestion, and their morphology was observed. The third passage of cells were stained with rhodamine-phalloidin for cytoskeleton observation. The third passage of cells were identified with flow cytometry through the detection of expressions of cell surface markers CD29, CD31, CD34, CD90, CD105, SSEA3, SSEA4 and immunity-related marker human leukocyte antigen-D related site (HLA-DR). The third passage of cells were also assessed the ability of adipogenic and osteogenic differentiation. (2) The third passage of human amniotic epithelial stem cells were cultured in DMEM medium supplemented with 10% exosome-free fetal bovine serum. Exosomes were isolated from culture supernatant by the method of ultracentrifugation and represented with scanning electron microscope for morphologic observation. (3) Six adult SD rats were anesthetized, and four 1 cm×1 cm sized wounds with full-thickness skin defect were made on the back of each rat. The wounds on the back of each rat were divided into control group, 25 μg/mL exosomes group, 50 μg/mL exosomes group, and 100 μg/mL exosomes group according to the random number table (with 6 wounds in each group), and a total volume of 100 μL phosphate buffered saline, 25 μg/mL exosomes, 50 μg/mL exosomes, and 100 μg/mL exosomes were evenly injected around the wound through multiple subcutaneous sites, respectively. The wound healing rate was calculated based on measurement on post injury day (PID) 7, 14, and 21. On PID 21, the healed wound tissue of each group was collected and stained with HE to observe and count skin accessories, and the arrangement of collagen fibers was observed with Masson staining. Data were processed with analysis of variance for repeated measurement, analysis of variance of randomized block design, one-way analysis of variance, and Bonferroni test. Results: (1) The cells, which were isolated and cultured, displayed typical cobblestone morphology with many microvilli on cell surface. Among the cells, the positive expression rates of CD29, CD90, SSEA3, and SSEA4 were above 50.0%, and the rate of CD105 was 8.0%, while the rates of CD31, CD34, and HLA-DR were almost 0. The cells could differentiate into adipocytes and osteoblasts. The above results revealed that the cells cultured were human amniotic epithelial stem cells. (2) Human amniotic epithelial stem cells-derived exosomes were round or oval vesicles with diameter from 50 to 150 nm. (3) On PID 7 and 21, wound healing rates of the four groups were close (with P values above 0.05). On PID 14, wound healing rates of 50 and 100 μg/mL exosomes groups were (89.8±4.3)% and (92.0±4.6)% respectively, significantly higher than the wound healing rate of control group [(80.3±6.4)%, P<0.05 or P<0.01]. Moreover, the wound healing rate of 100 μg/mL exosomes group was significantly higher than that of 25 μg/mL exosomes group [(83.3±5.1)%, P<0.05]. On PID 21, the numbers of skin accessories in 50 and 100 μg/mL exosomes groups were 4.3±1.4 and 5.1±1.6 respectively, obviously more than those of control group and 25 μg/mL exosomes group (respectively 1.4±0.5 and 1.8±0.6, with P values below 0.01). Well reorganized collagen fibers were observed just in the healed wound tissue of 50 and 100 μg/mL exosomes groups. Conclusions: Human amniotic epithelial stem cells-derived exosomes can promote healing of wound with full-thickness skin defect in rats.

Effect of hydrocinnamoyl-L-valyl pyrrolidine on healing quality of deep partial-thickness scald wound in mice

Effect of hydrocinnamoyl-L-valyl pyrrolidine on healing quality of deep partial-thickness scald wound in mice

Effects of rabbit adipose-derived mesenchymal stem cells on the healing of skin deep partial-thickness scald wound of rabbit

To investigate the effects of local injection of rabbit adipose-derived mesenchymal stem cells (ADSCs) on the healing of skin deep partial-thickness scald wound of rabbit. ADSCs were isolated from adipose tissue of one New Zealand rabbit and then sub-cultured. ADSCs of the third passage were used in the following experiments. Twenty-four rabbits were divided into ADSCs group (n=12) and control group (n=12) according to the random number table, and one deep partial-thickness scald wound with diameter of 5 cm on the two sides of the back near the buttocks was made. From post injury day (PID) 2, 2 mL suspension of EdU-labeled ADSCs with the number of 5×10(5) per mL was subcutaneously injected in wounds of rabbits in ADSCs group, while the rabbits in control group were given 2 mL serum-free DMEM until the wounds were healed. Wound healing processes of rabbits in two groups were observed every day, and the healing time was recorded. On PID 7, 14, 21, and 28, areas of wound of three rabbits in two groups were measured and the healing rates were calculated, respectively. The healed wound tissue was harvested to observe the morphology by HE staining, and the expression of collagen fiber was observed by Masson staining. The distribution of EdU-labeled ADSCs in healed wound tissue on PID 28 was observed by inverted fluorescence microscope. The expressions of vascular endothelial growth factor (VEGF) and epidermal growth factor (EGF) of healed wound tissue on PID 7, 14, and 21 were detected by enzyme-linked immunosorbent assay. Data were processed with analysis of variance of factorial design and paired samples t test. (1) The wound healing time of rabbits in ADSCs group was (19.5±1.1) d post injury, which was significantly shorter than that in control group [(23.3±1.5) d, t=4.50, P<0.05]. On PID 7, wounds of rabbits in two groups were dry with no obvious exudation, and redness and swelling around wounds disappeared gradually, the wound healing rate of rabbits in ADSCs group was (15.1±2.4)%, which was close to that in control group [(13.7±3.1)%, t=1.20, P>0.05]. On PID 14, wounds of rabbits in ADSCs group were dry and scabbed obviously, and the wound healing rate was (73.1±5.7)%, while wounds of rabbits in control group were little scabbed with little exudation, and the wound healing rate was significantly lower than that in ADSCs group [(52.9±5.1)%, t=8.06, P<0.01]. On PID 21, wounds of rabbits in ADSCs group were generally healed, and the wound healing rate was (95.6±3.0)%, while a few wounds still existed in rabbits of control group, and the wound healing rate was significantly lower than that in ADSCs group [(78.6±3.7)%, t=9.73, P<0.01]. On PID 28, wounds of rabbits in two groups were totally healed with the healing rate of 100%, and texture and microvascular responses of healed wound tissue in ADSCs group were better than those in control group. (2) On PID 7, fibroblasts in healed wound tissue of rabbits in two groups were all increased, and there were little vascular and collagen fiber proliferation with no obvious differences. On PID 14, the number of fibroblasts in healed wound tissue of rabbits in ADSCs group was more than that in control group, and the collagen fibers in healed wound tissue of rabbits in ADSCs group were arranged in dense and uniform, while those in control group were sparse and irregular. On PID 21, skin layers were differentiated in healed wound tissue of rabbits in two groups, and collagen fibers in healed wound tissue of rabbits in ADSCs group were still denser than that in control group. On PID 28, newborn skin was well differentiated in healed wound tissue of rabbits in ADSCs group, which was better than that in control group. There were a lot of thick collagen fibers in healed wound tissue of rabbits in two groups, and EdU-labeled ADSCs were involved in skin texture of rabbits in ADSCs group. (3) The expressions of VEGF and EGF in healed wound tissue of rabbits in two groups were similar on PID 7 (with t values respectively 0.70 and 0.91, P values above 0.05), which in ADSCs group were significantly higher than those in control group on PID 14 and 21 (with t values from 2.85 to 4.81, P values below 0.01). The transplantation of ADSCs can promote the wound healing of skin deep partial-thickness scald wound of rabbit and shorten the wound healing time.

A systematic and quantitative method for wound-dressing evaluation.

BackgroundFor patients with skin defects such as burns, wound dressing plays important roles in protecting the wound. Before a novel wound dressing is applied to a patient, a series of tests should be performed to ensure its safety and efficacy. Different types of animal wound-healing models have been used to study the bio-function of different wound dressings; however, a systematic way to evaluate the effect of a wound dressing on wound healing and cutaneous regeneration is lacking.MethodsIn the study presented here, full-thickness wound models were established in mice, and a systematic way to quantitatively analyze the wound-healing process and the histological results is described.ResultsIt was found that the rate of wound healing in the tested wound dressing (TWD) group was higher than that in the control group, and the re-epithelialization and the formation of granulation tissue were enhanced when the TWD was applied. Meanwhile, the inflammatory response was attenuated in the TWD group, and more mature and better aligned collagen fibers in the healed wound tissue was found in the TWD group compared with that in the control group.ConclusionsA systematic, quantitative way to analyze the effect of a wound dressing on wound healing was established. And it might be helpful for the design of wound dressing in the future.

Cells from the hematopoietic lineage are only present transiently during healing in a mouse model of non-severe burn injury.

IntroductionThe aim of our study is to identify the contribution of hematopoietic-derived cells to burn-wound healing in a non-severe injury. There are many conflicting reports of the contribution of bone marrow-derived cells to wound healing and whether these are hematopoietic or mesenchymal in origin. The role of hematopoietic lineage cells is investigated in this study in the context of the response to burn injury.MethodsTransgenic mice expressing the LacZ reporter gene in all cells of the hematopoietic lineage underwent a non-severe full-thickness burn injury (8 % of total body surface area). Wounds were assessed for LacZ-positive cells at days 7, 14, and 28 post-injury by using whole-mount staining. Cells were also cultured from the wounds at each time point and analysed for expression of fibroblast and myofibroblast markers.ResultsAt day 7, positive cells were identified in the wounds representing the inflammatory response. Some dermal cells were also identified at this early stage. At day 14, positive cells were also identified and were cultured from the wound tissue samples. However, by day 28, no positive cells could be detected or cultured from the healed wound tissue. Isolated LacZ-positive cells did not express collagen 1 or α-smooth muscle actin proteins, indicating that they had not differentiated into dermal fibroblast-type cells.ConclusionsIn this model of burn injury, hematopoietic lineage cells were present in the healing wound only transiently and did not appear to contribute to the long-term scar population. This is in contrast with reports demonstrating that fibrocytes contribute a long-term sustained population in scar tissue. This work demonstrates that in a non-severe burn injury model there is a sustained transient contribution of hematopoietic cells to the healed wound. Further characterisation of the types and extent of wounding required to establish a long-term hematopoietic response will be important in determining future cell-based therapies.

A modified collagen gel enhances healing outcome in a preclinical swine model of excisional wounds

Collagen-based dressings are of great interest in wound care. However, evidence supporting their mechanism of action is scanty. This work provides first results from a preclinical swine model of excisional wounds, elucidating the mechanism of action of a modified collagen gel (MCG) dressing. Following wounding, wound-edge tissue was collected at specific time intervals (3, 7, 14, and 21 days postwounding). On day 7, histological analysis showed significant increase in the length of rete ridges, suggesting improved biomechanical properties of the healing wound tissue. Rapid and transient mounting of inflammation is necessary for efficient healing. MCG significantly accelerated neutrophil and macrophage recruitment to the wound site on day 3 and day 7 with successful resolution of inflammation on day 21. MCG induced monocyte chemotactic protein-1 expression in neutrophil-like human promyelocytic leukemia-60 cells in vitro. In vivo, MCG-treated wound tissue displayed elevated vascular endothelial growth factor expression. Consistently, MCG-treated wounds displayed significantly higher abundance of endothelial cells with increased blood flow to the wound area indicating improved vascularization. This observation was explained by the finding that MCG enhanced proliferation of wound-site endothelial cells. In MCG-treated wound tissue, Masson's trichrome and picrosirius red staining showed higher abundance of collagen and increased collagen type I:III ratio. This work presents first evidence from a preclinical setting explaining how a collagen-based dressing may improve wound closure by targeting multiple key mechanisms. The current findings warrant additional studies to determine whether the responses to the MCG are different from other collagen-based products used in clinical setting.

Efficacy of Jasminum grandiflorum L. leaf extract on dermal wound healing in rats.

Wound healing is a fundamental response to tissue injury and natural products accelerate the healing process. Here, we have explored the efficacy of topical administration of an ointment, prepared by methanolic extract of Jasminum grandiflorum L. (Oleaceae) leaves, on cutaneous wound healing in rats. The topical application of the Jasminum ointment on full thickness excision wounds accelerated the healing process. Tissue growth and collagen synthesis were significantly higher determined by total hydroxyl proline, hexosamine, protein and DNA content. The response was concentration- and time-dependent, when observed on days 4, 8 and 12 after wound creation. The rate of wound healing was faster as determined by wound contraction, tensile strength and other histopathological changes. In addition, this ointment also raised the activity of superoxide dismutase (SOD) and catalase (CAT) with high GSH content and low lipid peroxidation products in wound tissue. Thus, it could be suggested that the ointment from the methanolic extract of J. grandiflorum leaf improves the rate of wound healing by enhancing the rate of collagen synthesis and also by improving the antioxidant status in the newly synthesised healing wound tissue.

Abstract 992: Novel Role of Copper-dependent Transcription Factor Antioxidant-1 in Cell Proliferation and Wound Healing

Copper plays a fundamental role in regulating cell proliferation involved in wound healing and angiogenesis. Recently, we found that antioxidant-1 (Atox1), previously appreciated as a copper chaperone at the transGolgi network (TGN), stimulates cyclin D1 transcription and cell proliferation in a copper dependent manner. However, functional role of Atox1 in wound healing in vivo remains unknown. Here we show that Atox1 was highly expressed at the BrdU positive nuclei in the wounding tissue, which was associated with an increase in copper content (2.1-fold), as assessed by ICP-mass spectrometry, as compared to non-wounded skin. In Atox1 knockout (KO) mice, the rate of wound closure was markedly delayed as compared to wild-type (WT) mice (7.1 day vs. 11.2 day for complete wound closure in WT vs. Atox1 KO mice, p &lt; 0.001), and BrdU-positive proliferating cells and cyclin D1 protein expression were significantly decreased. Capillary density in healing wound tissue was also reduced in Atox1 KO mice (27.8% decrease). To segregate a copper-dependent transcription factor and a copper chaperone function of Atox1, we have generated lentivirus expressing nuclear targeted Atox1 (Atox1-NLS), TGN-targeted Atox1 (Atox1-TGN) or Atox1-WT. and examine their effect on cell proliferation and wound healing in vitro and in vivo. In cultured Atox1 KO mouse fibroblasts, copper-induced cell proliferation and cyclin D1 expression were abolished, which was rescued by re-expression of both Atox1-NLS and Atox1-WT, but not Atox1-TGN. Promoter deletion analysis identified Atox1 responsive element (Atox1-RE) at cyclin D1 promoter and EMSA and ChIP assays demonstrated that Atox1 binds to Atox1-RE at the cyclin D1 promoter in a copper dependent manner. The impairment of wound healing in Atox1 KO mice was rescued by re-expression with lenti-Atox1-NLS and Atox1-WT, but not Atox1-TGN, which was associated with the increase in cyclin D1 expression as well as number of BrdU positive proliferating nuclei. In summary, transcription factor function of Atox1, but not copper-chaperone function of Atox1, plays a critical role in copper-induced cell proliferation, which may contribute to wound repair process in response to injury in vivo.

A non-invasive, three-dimensional, diagnostic laser imaging system for accurate wound analysis

A PC-based laser imaging system has been developed that can accurately image and analyse three-dimensional (3D) surfaces in healing wound tissue. The device consists of a laser diode and sensor mounted to a motorized, bi-directional, X - Y table. The laser measures the distance to the wound surface as the X - Y table moves over the wound. The laser beam diameter is and can measure distances as small as . The distance information from the laser is used to produce the 3D image and to calculate the volume of the wound. The accuracy of this device is demonstrated by measuring rubber model wounds of 0.5, 1 and 2 ml. The per cent error for the 0.5 and 1 ml model wounds was less than 1 per cent and approximately 4 per cent for the 2 ml model. Studies were done in which full-thickness wounds were made on rats and monitored over a two week period. The rats were scanned five times during this period and the wound area and volumes calculated. The laser measurements are compared to measurements using saline and wound thickness multiplied by wound area. Keywords: volume, surface area, non-invasive, laser imaging, wound healing

Differential Expression of Heat Shock Protein 70 in Well Healing and Chronic Human Wound Tissue

Heat shock protein 70 (hsp 70) is an important member of the heat shock protein family, which is induced by different forms of stress. We attempted to find out if hsp 70 is also involved in wound healing, which likewise resembles a stress situation for cells too. Therefore we collected tissue samples from well healing and chronic human wound tissue. We used Northern- and Western-blot analysis to study the expression of hsp 70. At the protein level we found a strong correlation between well healing wounds and high expression of hsp 70, whereas chronic wounds showed no or weak expression. Interestingly hsp 70 mRNA did not show this significant correlation, displaying a variant expression pattern in the same kind of wound tissue, possibly due to an unknown posttranscriptional regulating step, which has to be investigated in further studies. To localize hsp 70 mRNA and protein we used in situ hybridization and immunohistochemistry. Both displayed an overexpression in endothelial cells of capillary vessels.

Healing of periodontal connective tissues following surgical wounding and application of citric acid in dogs.

Topical application of citric acid to a root surface which has been scaled and root planed results in exposure of collagen fibrils of the dentin matrix. The purpose of this study was to examine the healing of gingival connective tissue adjacent to a root planed and subsequently acid conditioned root surface and to determine whether the exposure of dentin or cementum matrix collagen would facilitate the reestablishment of an attachment between the healing connective tissue and the root surface. Full thickness flaps were raised and labial alveolar bone removed over maxillary incisor roots of seven Labrador retrievers. The exposed root surface was planed before etching with citric acid (pH 1) for 3 min. The flap was then repositioned at its original level. Block sections were obtained after 7, 14, 21 and 42 days and the interface between the root surface and the healing wound tissue studied by light and electron microscopy. Compared to conventional flap surgery in the same animals, application of citric acid to the instrumented root surface resulted in an improved rate of connective tissue healing as well as a rapid and consistent reestablishment of connective tissue attachment through extensive interdigitation of new and old collagen fibrils at the tooth‐gingiva interface. Subsequent reinforcement of the established connective tissue attachment included recalcification of the acid‐affected dentin and deposition of new cementum.

Uptake of Radiosulfur During the Healing of Surgical Wounds

Abstract The feasibility of using the deposition of S35 in wounds as a measure of the rate of healing was investigated by administering isotopically labeled methionine, cystine, or sulfate to surgical patients. It was found that the level of S35 measurable at the surface of the wounds was higher when either methionine-S35 or cystine-S35 was given after wounding than when given before. More S35 was incorporated into the wound tissue when methionine was the source of the isotope than when labeled cystine supplied the S35. About 4-7 times as much S35 was detectable in the regenerating wound tissue within a few days after wounding, compared to S35 detectable in unaltered skin. Only a relatively small amount of sulfate-S35 was taken up by healing wound tissue. These results were obtained by the use of a technic which made it possible to measure repetitively, over a long period, the activity at the surface of the same sample of wound tissue.

Local increase of poliomyelitis virus in healing wound tissue of cynomolgus monkeys.

Local increase of poliomyelitis virus in healing wound tissue of cynomolgus monkeys.