| There are about 5 to 10 million people suffering from burn in China annually. Due to the long period of treatment and recovery, the outcome of the patients was very poor with a high mortality rate, face damage rate and mutilation rate. Therefore, it is critical to develop novel strategies for initial precision diagnosis and accurate management.For the diagnosis of burn skin, the accurate judgment rate of burn depth only reaches 60 -75% with patients' clinical manifestation and doctors' experience. Thus, various techniques for auxiliary diagnosis were developed, including ultrasonography, MRI,vascular microscope, fluorescence staining, THZ and multi spectrum. However, most of these techniques were based on the traditional three degree four points methods, which limited in detecting the exact depth of the necrotic tissue. Though the pathological technique provides the method for examining, it is just an invasive procedure and causes permanent damage. So far there is no non-invasive method to detect the burn depth accurately. Thus, novel techniques for accurate detection, which will provide us a new classification method of burn depth, as well as new insight into precision debridement, were emergent for burn patients.For the treatment of burns, skin wounds exposed to air must be covered with a dressing, as a barrier, to play a temporary protective effect. There are for two problems, one of them is that when is the right time to replace the wound dressings. Burn wound dressing has been relying on the experience of the clinician, so far there are no objective evaluation standards for precise told doctors when to replace the dressing. It is a positive sign that when the wound fluid completely soaked in medical dressings to remind the doctor must immediately change the dressing. Unnecessary dressing changing wastes manpower and resources, and delayed dressing changing increases the risk of infection. Thus, a more accurate and prompt evaluation of bleeding or exudates is indispensable for surgery.Another problem is that how to choose and make new wound dressings as the clinical dressing varied each other's. In recent years, many kinds of wound dressings have been developed, and the tissue engineering dressings of the synthetic dressings have been developed in recent years. In particular, with the gradual in-depth study of the electrostatic spinning technology, the development of the electrostatic spinning fiber membrane dressing has become a new hot spot for the research of the skin wound dressing. Because of the limitation of the electro spun fibrous membrane characteristics, electrostatic spinning fiber membrane dressing does not contain a healing factor. Through publicly available reports of electro spun fibrous membrane, the dressing of the wound healing factor of the electrostatic spinning fiber membrane is complicated and cannot be released slowly which caused the poor effect of promoting healing and the difficulty of clinical transformation. Therefore, if we can make the wound dressing slowly releasing the promoting factor on the electrostatic spinning fiber film through the simple method, and prove the effect and mechanism of the materials on wound healing at the same time, so we can provide a new idea for clinical selection and production of this new type of wound dressing, enrich the theoretical basis of wound dressing to promote wound healing, and lay a solid foundation for the next clinical transformation.Therefore, this study consists of three parts. In the first part, according to the study of near infrared spectroscopy technology, we want to accurately diagnose the depth of burn wound skin necrotic tissue which can improve the diagnostic accuracy of depth of burn wound. The significance lies in not only can provide a new idea of precise burn classification, but also can provide objective reference for clinicians to judge the burn depth which would lead to the increasing of the quality of burn wound healing; In the second part,we plan to evaluate the signal penetration capacity of THz-rays through gauze and the THz spectral differences among gauze samples containing different liquids.As well as the sensitivities of THz for detecting liquid-content in gauze which, to a certain extent, can reduce the pain of patients and save some medical and human resources. In the third part,we explore the effect of VIP-DA-PCL Nano fiber membrane on wound healing in vivo and its related mechanism, which could provide a new way of thinking about the selection and production of burn wound dressing, enrich the theoretical basis of the same type of dressing to promote wound healing, and lay the foundation for the next clinical transformation.Part 1: Near infrared spectroscopy used to accurately diagnose the depth value ofburn wound necrosis tissueObjective : Making clear the relationship between burn depth and burn time;Establishing The relationship between the original spectral data and the burn time;Characterizing the functional status of burn skin by extracting the information of component information and structural information of burn wound; Obtaining The depth inversion model and the accuracy of the model.Method:?Different burn rat model of different burn time was made. With the increase of burn time, it was subdivided into nine groups: 5S, 10s, 20s, 30s, 40s and 60s,80s,100s,120s,each group had five samples.?Opening the spectrometer to adjust it to the data collection status. Burn wound and incident optical fiber maintains vertical. The distance of the fiber probe and surface is 2cm,?After collecting the spectrum data on wound, we harvested immediately to obtain HE staining results. And the histological observation was measured at each burn time points which corresponding to specific burn depth value. Each specimen was measured five times and depth values were calculated by two independent pathological workers.?Collected spectral information data were analyzed and the optical parameters were extracted. Using Monte Carlo analysis, extraction of the component information (hemoglobin, hemoglobin, water) and structural information(scattering particle size and concentration) were used to characterize the functional status of burn skin;?The obtained optical characteristic parameter samples are used for support vector machine (SVM) training. 1557 total samples are divided into two parts. The first part is 2/3 (1037 samples) of the total sample, which is utilized to build and train the support vector machine model. Training methods is cross validation method, digging out the relationship of optical characteristics and the burn which is linear or nonlinear. Establishing the complex mapping relation model of burn skin tissue and composition structure; then the second part is the 1/3 (520 samples) of the total sample, and the model was used to carry out the 10 inversion to verify the effectiveness of the model.Results: ?A linear model was established between the burn time and the burn depth.Under the control of the burn time, the depth of the scale increased monotonously with the increase of the burn time in 1-120s.?We got the relationship between the original near infrared spectral data and scalding time,and found that with the increase in the burn time,the spectral amplitude of the spectral curve in the specific band (900-1300nm) gradually reduced. And with the increasing of the depth of burn, the spectral curves gradually tend to be smooth.?The optimal feature set with five parameters is extracted, and the parameters are regular, but the law is not obvious.?The SVM was successfully used to construct the model of the inversion of a deep burn. And according to 10 times in the inversion of the model, the average relative error of the mean was 7.63%.Conclusion: Near infrared spectroscopy data can be obtained about the burn skin necrosis, and then using the procedure of Monte Carlo parameter extraction and the training of SVM parameters, the model of burn depth inversion can be constructed. Through this model, the exact depth value of the necrotic tissue in the burn wound can be accurately retrieved from the collected near infrared spectral data, and the average relative error of the mean was 7.63%. The diagnostic accuracy is in the range of hundred micrometers.Part 2: Precision measurement of various liquids in gauze using terahertz time-domain spectroscopyObjective: We evaluated the signal penetration capacity of THz-rays through gauze to explore the possibility of the terahertz spectrum for the detection of the effusion in gauze.Then we evaluated the THz spectral differences among gauze samples containing different liquids.As well as the sensitivities of THz for detecting liquid-content in gauze to confirm that the accurate diagnosis of the content of the exudates in the gauze can be accomplished by the terahertz spectrum.Methods:?The gauze used in the experiment was regular dry medical (cotton) gauze with a cotton fiber diameter of 0.34 mm and a 1 × 1 mm fiber interval,as determined using a Vernier caliper The dimensions of the gauze pieces used for the experiments were 5×5 cm. Unusual numbers of layers (from 1 to 80 layers) were fixed to a holder for the detection of the THz-TDS penetration capacity.; ?The linear relationship between the THz wave transmission characteristics and the layer number is obtained by the formula derivation and simplified formula; ?A volume of 1000 ml of whole blood was collected from an adult pig,and 0.05 ml of 8% trisodium citrate was added to 1 ml of blood to prevent clotting. Half of the blood immediately stored in a blood collection tube, and the remainder was centrifuged at 2000 rpm for 30 min to remove the plasma from the RBCs. The plasma was removed,and the RBCs were discarded. The water that was used for testing wasdeionized water produced by Millipore. First,5 × 5cm gauze sample of 40 layers in thickness was subjected to THz-TDS to obtain a reference light signal. Then, a syringe was used to drop various total amounts of each sample liquid (demonized water, plasma or whole blood) onto the centers of additional gauze samples, which were allowed to stand for 5 minutes to allow for the osmosis of the liquid and were then subjected to THz-TDS.?Under the assumption that the sample liquid is distributed homogeneously in the gauze, the total content of the sample liquid in the medical gauze can be calculated by simplifying the experimental model and formula, then the corresponding model between the total content of sample liquid and the THz reflectivity at 0.3T in gauze was obtained.Results: ?The results of this experiment demonstrated that THz radiation exhibited excellent penetration through up to 80 layers of dry gauze. The natural logarithm of the THz power transmittance decreased with an increasing number of gauze layers.?The results indicated that the THz absorbance monotonously increased with increasing liquid content in the gauze and that different types of liquids exhibited different THz absorbencies.For a given liquid content in the gauze, the highest THz absorbance was observed for whole blood, followed by plasma anddeionized water. The slope of the fitting curve of the three liquids was 0.1518 for deionized water, 0.089 for whole blood, and 0.0684 for plasma;these values were used to estimate the measurement sensitivities for the three liquids.?The sensitivity was 1.3mg for deionized water, 0.76mg for whole blood and 0.58mg for plasma.These results indicate that any change in the deionized water content in a gauze sample of greater than 1.3mg could be detected using THz-TDS.Conclusion: THZ wave of medical gauze with good penetration performance, can be very accurately detect the content of three kinds of liquid in gauze (whole blood, plasma and water), and the detection sensitivity of this level is more than 1.3mg.Part 3: Study on VIP-DA-PCL Nano fiber membrane for promoting wound healingObjective: VIP-DA-PCL Nano fibrous membrane material loaded with vasoactive intestinal peptide in vivo was studied to make clear the effect of the material on wound healing; through the localization and quantification of cell proliferation markers in the wound tissue, and the quantitative detection of angiogenesis markers to make clear the effect of the material on cell proliferation and vascular regeneration.Method: ?For anesthesia, 1% pentobarbital were intraperitoneally injected into the BALB/c mice (0.01 mg/g of body weight). The dorsal surface was carefully shaved,sterilized with 75% alcohol and then punched to full-thickness defect wounds with a diameter of 4mm. The sterilized Vaseline gauze, PCL-DA and PCL-DA-VIP Nano sheets were implanted into each wound, respectively. All the wounds were then covered with a biological membrane.Wounds covered with only the biological membrane served as the control groups.?As wound healing was generally measured by the closed area of wounds,we therefore photographed the wounds at day 1, 3, 5 and 7 post-surgery and used IPP 6.0 software to measure the closed wound area by two independent researchers.?Hematoxylin-eosin (H&E) staining was used to detect the length of the newly-regenerated epidermis, which was defined as the distance between the leading edges of the newly-regenerated epidermis and the edges of unwounded epidermis Because some wounds in PCL-DA-VIP group have been completely epithelialized at day 7 post-surgery,the length of the newly-regenerated epidermis was measured only at day 3 post-surgery by H&E staining.?Masson trichrome staining was utilized to detect the thickness of the granulation tissue at day 3 and day 7 post-surgery,respectively.?To detect the proliferation rate of keratinocytes in the newly-regenerated epidermis, the proliferating cell nuclear antigen (PCNA), which is a widely used cell proliferation marker, was immunohistochemically stained by day 3 post-surgery.?To further quantitatively detect the cell proliferation rate and the vascularization, the proliferation maker (PCNA) and the antigenic markers (CD31)and vascular endothelial growth factor (VEGF) in the full-thickness wound tissues were tested by Western Blotting at day 3 post-surgery, respectively.Results: ?Wound healing in the PCL-DA-VIP group was found significantly faster than that of other groups (p < 0.05). At day 7 post-surgery, 96.5% of the wound area in PCL-DA-VIP group had been closed. Meanwhile, the percentage of closed wound area in control group, Vaseline gauze group and PCL-DA group were 89.25%, 83.25% and 88.25%(p < 0.05);?The length of the newly-regenerated epidermis in both PCL-DA group and PCL-DA-VIP group was found significantly longer than that in control group and Vaseline gauze group at day 3 post-surgery(p < 0.05);However, no significant difference of the length of the newly-regenerated epidermis was observed between the PCL-DA group and PCL-DA-VIP group.?The thickness of granulation tissue in the PCL-DA-VIP group was significantly larger than that in other groups at both day 3 and day 7 post-surgery (day 3, p< 0.01; day 7, p < 0.001).?The PCNA positive keratinocytes in both the PCL-DA group and the PCL-DA-VIP group were found more than that in the control group. However, no significant difference of cell proliferation in the newly-regenerated epidermis was observed between the PCL-DA group and PCL-DA-VIP group. In the Western blot experiment, the optical density values of PCNA bands of the PCL-DA-VIP group were significantly higher than those of other groups Western blotting (p < 0.001)??The CD31 and VEGF proliferation in the full-thickness wound tissues was detected by Western blotting. The optical density vallues of CD31 bands of the PCL-DA-VIP group were significantly higher than those of other groups (p < 0.05). The optical density values of VEGF bands of the PCL-DA-VIP group were significantly higher than those of the control group and the PCL-DA group (p < 0.05)? However, no significant difference of VEGF was found between the Vaseline gauze group and PCL-DA-VIP group.Conclusion: PCL-DA-VIP Nano fiber membrane can promote the healing of wounds in vivo, which also can promote the growth of granulation tissue and angiogenesis, but has no significant improvement in the re - epithelium of the wound.General conclusion: Based on the above three aspects, we draw the following conclusions through the research on the diagnosis and treatment of burns:1. Near infrared spectroscopy can accurately measure the depth value of burn skin necrosis, the average error of the model of burn depth inversion error was 7.63%. The diagnostic accuracy is in the range of hundred micrometers.2. THZ waves of medical gauze with good penetration performance, can be very accurately detect the content of three kinds of liquid in gauze (whole blood, plasma and water), and the detection sensitivity of this level is more than 1.3mg.3. PCL-DA-VIP Nano fiber membranes can promote the healing of wounds in vivo,which also can promote the growth of granulation tissue and angiogenesis, but has no significant improvement in the re - epithelium of the wound.The realization of the above goals could provide some new ideas, new methods for the diagnosis and treatment of burn; preliminary exploration on the aspects of mechanism would also provide a certain theory and data support for the precise medical development of burn. |
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