[Preparation of chitin/hyaluronic acid/collagen hydrogel loaded with mouse adipose-derived stem cells and its effects on wound healing of full-thickness skin defects in rats].

Objective: To prepare the chitin/hyaluronic acid/collagen hydrogel loaded with mouse adipose-derived stem cells and to explore its effects on wound healing of full-thickness skin defects in rats. Methods: The research was an experimental research. Chitin nanofibers were prepared by acid hydrolysis and alkaline extraction method, and then mixed with hyaluronic acid and collagen to prepare chitin/hyaluronic acid/collagen hydrogels (hereinafter referred to as hydrogels). Besides, the hydrogels loaded with mouse adipose-derived stem cells were prepared. Thirty male 12-week-old guinea pigs were divided into negative control group, positive control group, and hydrogel group according to the random number table, with 10 guinea pigs in each group. Ethanol, 4-aminobenzoic acid ethyl ester, or the aforementioned prepared hydrogels without cells were topically applied on both sides of back of guinea pigs respectively for induced contact and stimulated contact, and skin edema and erythema formation were observed at 24 and 48 h after stimulated contact. Adipose-derived stem cells from mice were divided into normal control group cultured routinely and hydrogel group cultured with the aforementioned prepared hydrogels without cells. After 3 d of culture, protein expressions of platelet-derived growth factor-D (PDGF-D), insulin-like growth factor-Ⅰ (IGF-Ⅰ), and transforming growth factor ß1 (TGF-ß1) were detected by Western blotting (n=3). Eight male 8-week-old Sprague-Dawley rats were taken and a circular full-thickness skin defect wound was created on each side of the back. The wounds were divided into blank control group without any treatment and hydrogel group with the aforementioned prepared hydrogels loaded with adipose-derived stem cells applied. Wound healing was observed at 0 (immediately), 2, 4, 8, and 10 d after injury, and the wound healing rate was calculated at 2, 4, 8, and 10 d after injury. Wound tissue samples at 10 d after injury were collected, the new tissue formation was observed by hematoxylin-eosin staining; the concentrations of interleukin-1α (IL-1α), IL-6, IL-4, and IL-10 were detected by enzyme-linked immunosorbent assay method; the expressions of CD16 and CD206 positive cells were observed by immunohistochemical staining and the percentages of positive cells were calculated. The sample numbers in animal experiment were all 8. Results: At 24 h after stimulated contact, no skin edema was observed in the three groups of guinea pigs, and only mild skin erythema was observed in 7 guinea pigs in positive control group. At 48 h after stimulated contact, skin erythema was observed in 8 guinea pigs and skin edema was observed in 4 guinea pigs in positive control group, while no obvious skin erythema or edema was observed in guinea pigs in the other two groups. After 3 d of culture, the protein expression levels of PDGF-D, IGF-I, and TGF-ß1 in adipose-derived stem cells in hydrogel group were significantly higher than those in normal control group (with t values of 12.91, 11.83, and 7.92, respectively, P<0.05). From 0 to 10 d after injury, the wound areas in both groups gradually decreased, and the wounds in hydrogel group were almost completely healed at 10 d after injury. At 4, 8, and 10 d after injury, the wound healing rates in hydrogel group were (38±4)%, (54±5)%, and (69±6)%, respectively, which were significantly higher than (21±6)%, (29±7)%, and (31±7)% in blank control group (with t values of 3.82, 3.97, and 4.05, respectively, Pvalues all <0.05). At 10 d after injury, compared with those in blank control group, the epidermis in wound in hydrogel group was more intact, and there were increases in hair follicles, blood vessels, and other skin appendages. At 10 d after injury, the concentrations of IL-1α and IL-6 in wound tissue in hydrogel group were significantly lower than those in blank control group (with tvalues of 8.21 and 7.99, respectively, P<0.05), while the concentrations of IL-4 and IL-10 were significantly higher than those in blank control group (with tvalues of 6.57 and 9.03, respectively, P<0.05). The percentage of CD16 positive cells in wound tissue in hydrogel group was significantly lower than that in blank control group (t=8.02, P<0.05), while the percentage of CD206 positive cells was significantly higher than that in blank control group (t=7.21, P<0.05). Conclusions: The hydrogel loaded with mouse adipose-derived stem cells is non-allergenic, can promote the secretion of growth factors in adipose-derived stem cells, promote the polarization of macrophages to M2 phenotype in wound tissue in rats with full-thickness skin defects, and alleviate inflammatory reaction, thereby promoting wound healing.

Inhibitory effect of pulmonary surfactant proteins A and D on allergen-induced lymphocyte proliferation and histamine release in children with asthma.

The role of pulmonary surfactant proteins in the pathogenesis of airway inflammation and the impact on asthma has not been elucidated. This study was designed to examine the effect of surfactant proteins A (SP-A) and D (SP-D) on phytohemagglutinin- (PHA) and mite allergen Dermatophagoides pteronyssinus (Der p)-induced histamine release and the proliferation of peripheral blood mononuclear cells (PBMC) in children with asthma in stable condition (n = 21), asthmatic children during acute attacks (n = 9), and age-matched control subjects (n = 7). The results show that SP-A and SP-D were able to reduce the incorporation of [3H]thymidine into PBMC in a dose-dependent manner. In addition to the intact, native SP-A and SP-D proteins, a recombinant peptide composed of the neck and carbohydrate recognition domain (CRD) of SP-D [SP-D(N/CRD)] was also found to have the same suppressive effect on lymphocyte proliferation. This effect was abolished by the presence of 100 mM mannose (for SP-A) or maltose (for SP-D) in the culture medium, which suggested that the CRD regions of SP-A and SP-D may interact with the carbohydrate structures on the surface molecules of lymphocytes. The inhibitory effects of surfactant proteins on PHA- and Der p-stimulated lymphocyte responses were observed in stable asthmatic children and age-matched control subjects, while only a mild suppression (< 25%) was seen in activated lymphocytes derived from asthmatic children with acute attacks. SP-A and SP-D were also found to inhibit allergen-induced histamine release, in a dose-dependent manner, in the diluted whole blood of asthmatic children. We conclude that both SP-A and SP-D can inhibit histamine release in the early phase of allergen provocation and suppress lymphocyte proliferation in the late phase of bronchial inflammation, the two essential steps in the development of asthmatic symptoms. It appears that SP-A and SP-D may be protective against the pathogenesis of asthma.