| ObjectiveTo construct a wound repair material with properties of hemostasis,anti-infection and promoted wound healing.Its physicochemical properties,biological safety,hemostatic properties,antibacterial properties and promoted wound healing effects were studied in vitro and in vivo,in order to provide a new and practical dressing material for wound healing of war injuries.Method1.The CuNPs/CA/CMC sponge with different concentration of CuNPs was prepared by freeze-drying method.Scanning electron microscopy（SEM）,fourier transform infrared spectroscopy（FT-IR）,X-ray diffraction（XRD）and liquid replacement method were used to observe the physicochemical properties and characterization of sponges.2.The cell proliferation was analyzed by CCK-8 and live/dead staining.The concentration of CuNPs with good biocompatibility was selected.The influence of sponge on blood coagulation was evaluated by the adhesion experiment of red blood cell,the blood absorption rate and hemostasis in vitro.The antibacterial abilities on the Staphylococcus aureus（S.aureus）and Escherichia coli（E.coli）was studied by plate colony-counting method,and the migration effect of sponge was studied by scratch test.3.The hemostatic ability of sponge was evaluated by rat-tail amputation model.The calculation of wound area and the histomorphology analysis was used to evaluate the wound healing and biological safety of sponge for the infected wound model.Results1.Both CA/CMC and CuNPs/CA/CMC sponges have porous structures.CuNPs/CA/CMC sponge has regular distribution and smooth boundary pores,which was 79.8%in porosity.The peaks of Alg and CMC at 1597 cm-1 and 1608 cm-1 were replaced by new peaks at 1602 and 1600 cm-1,indicating the polymer cross-linked by Ca2+ and Cu2+ions.Compared with CA/CMC sponge,the characteristic peak for carbonyl groups（-COOH and-C=O）in CuNPs/CA/CMC sponge were shifted and enhanced from 1600 to 1602 cm-1 and from 1422 to 1424 cm-1,respectively.Accordingly,the peak for the hydroxyl group（-OH）was also shifted and intensified from 3432 cm-1 to 3391 cm-1.The XRD spectrum indicated that most peaks were consistent with the standard cubic Ca2Cu crystal phases（JCRDS 22-0524）.2.The proliferative activity of CA/CMC group and 1 mmol/L CuNPs group was higher than 70%,and the HUVECs have normal morphology and the state of good growth,but 2 mmol/L CuNPs group and 4 mmol/L CuNPs group was lower than 70%,and the former of them was a small amount of living cells,which were round in shape,and the letter of them was only found a few cells in the edge of the plate,and most of which were dead cells.In interfacial interaction between the sponges and red blood cell（RBC）,the distribution of RBC was scattered in CA/CMC group and the CuNPs/CA/CMC group showed rouleaux formation of erythrocytes.The blood absorption capacity of CuNPs/CA/CMC sponge is higher than that of CA/CMC sponge and medical gauze.In blood coagulation experiment,hemoglobin concentration of gauze group was（34.77±7.23）g/L,while that of CA/CMC group was（166.45±11.38）g/L and that of CuNPs/CA/CMC group was（172.73±6.71）g/L.In antibacterial test,the antibacterial rates of CuNPs/CA/CMC sponge were（99.75±0.08）%and（99.86±0.04）%in S.aureus and E.coli,respectively,while CA/CMC sponge has little antibacterial ability.In migration assay,the scratches in CuNPs/CA/CMC group were completely closed at 36h but not in others.3.In hemostasis experiment,the hemostasis time of gelatin group（500.0±72.87）s and CuNPs/CA/CMC sponge group（398.8±65.41）s was shorter than that of gauze group（713.8±45.55）s,but there was no significant difference between the two groups in former.In infected wound healing,CuNPs/CA/CMC sponge with good biocompatibility was shorter than CA/CMC group in healing time,and improved the quality of regenerative skin and good biocompatibility in vivo.Conclusion1.CuNPs/CA/CMC sponge was successfully prepared by freeze-drying method.Its porous structure was observed by SEM.FT-IR and XRD suggested that CuNPs was cross-linked with sodium alginate and carboxymethyl cellulose.In addition,the measurement of porosity and swelling ratio suggest that CuNPs/CA/CMC sponge has high porosity and good swelling performance;2.The higher the concentration of copper,the stronger the cytotoxicity of sponge.The 1mmol/L CuNPs concentration,which has little cytotoxicity,was selected as the concentration of CuNPs in this study.CuNPs/CA/CMC sponges are more conducive to promoting the aggregation of RBC than CA/CMC sponges,so that the former have higher blood absorption rate than the latter,and coagulate more hemoglobin at the same time.That will be beneficial to the formation of larger clots and improve hemostasis.CuNPs/CA/CMC sponge has good antibacterial ability to S.aureus and E.coli,while CA/CMC sponge has little antibacterial ability.CuNPs/CA/CMC sponge can promote the migration of HUVECs more than CA/CMC sponge.3.The hemostasis ability of CuNPs/CA/CMC sponge is similar to that of gelatin,and it has a certain therapeutic effect on infected wound.It can accelerate wound healing,promote the formation of collagen fibers,and improve the quality of regenerative skin,and have good biocompatibility. |
