Comparative Analyses Of Histological And Material Properties Of Reticular Dermis Derived From Human, Swine And Rat

Background:Acellular dermal matrix is commonly used as dermal substitute to reconstruct and repair skin defect in clinical practice. Human acellular dermal matrix is the "gold standard" of repair and regeneration of dermal defects, but the limited sources of allogeneic skin, and higher product prices, limit its widespread application in clinical practice. Therefore, preparation of xenogenic acellular dermal matrix from animals is key to solving this problem. In recent years, researches for the acellular dermal matrix from pigs and rats have been a hot topic, but both material’s properties and their differences few report. Previous study in our lab found that the texture of porcine reticular dermis at lateral ventral part is soft, and after decellularized, the cell infiltration is good when co-cultured with fibroblasts. Therefore, this study will contrast porcine reticular dermis at lateral ventral part and rats reticular dermis at dorsal part with human reticular dermis in terms of histomorphology and material characterization to provide source choice basis for preparation of xenogenic acellular dermal matrix.Object:For species-source selection of xenogenic acellular dermal matrix, we compare lateral abdomen reticular dermis of pigs that were studied by my laboratory and rats reticular dermis with human reticular dermis though tissue morphology observation, biomechanics analysis, Fourier transform infrared spectroscopy scanning, thermal analysis, to further clarify material character differences of them in preparation of acellular dermal matrix, and provide scientific basis of selection of xenogenic acellular dermal matrix preparation materials.Methods:1. Experimental samples source:Normal full-thickness skin derived three adult males. Six white male pigs, four to five months old. Six clean grade male Wistar-rats, 9-week-old.2. Sample preparation:After unhairing, full-thickness skin specimen is cut from side abdominal part of each pig, and from backside of each Wistar-rat. Human full-thickness skin specimen is donated by three males. Reticular dermis is derived from all of the skin samples with drum dermatome. Reticular dermis specimens for histology staining are fixed with paraformaldehyde, paraffin-embeded conventionally, and cut into slices with histotome. The specimens for biomechanical test, FTIR spectroscopy scanning, thermal analysis are vacuum-freeze-dried.3. Procedures and observations:(1) Gross examination:compare the texture of the 3 groups of samples by macroscopic observation and touch with hand.(2) Biomechanical specimen making:the vacuum freeze-drying samples are cut into 80 mm × 5 mm, rehydrated, and then conduct uniaxial tensile tests. The test data are statistically analyzed.(3) Hematoxylin-eosin staining:Paraffin sections were hematoxylin-eosin stained conventionally, and take pictures under a light microscopy. In the pictures make the following measurements with image analysis software:① Measure the diameter of collagen fiber bundles.② Measure the percentage of the blank area in the entire picture area.(4) Picric acid and Sirius red staining:Tissue sections were stained with picric acid-Sirius red, and take pictures under a polarizing microscope. Type Ⅰ, Ⅲ collagen fiber content and their ratio in the pictures were measured with image analysis software..(5) Fourier transform infrared spectroscopy scanning:After vacuum-freeze-dried, the samples were prepared though Tablet Method, and undertake Fourier transform infrared spectroscopy scanning.(6) Thermal analysis:After vacuum-freeze-dried, the specimens were tested for thermogravimetric analysis curves and differential scan calorimetry curse with simultaneous thermal analyzer. Drawing software Origin 8.0 is used to make derivative thermogravimetric curve based on experimental results.Results:1. Gross examination:The porcine reticular dermis with densely big pore is slightly stiffer than humans’and it’s extensibility is poor. However, rat reticular dermis with smaller pores is delicate and softer. The human dermis with sparsely small pores has a bigger ductility and it’s texture is between the pig’s and rat’s.2. Biomechanical test:Three sets of rehydrated samples are good. Compared with human groups, the Young modulus difference was not statistically significant, porcine side abdomen dermis (P=0.44), rat back dermis (P=0.36).3. Hematoxylin-eosin staining:(1) Observation by light microscope:Collagen fiber bundles of porcine reticular dermis is thick and big, and their arrange is crowded; however collagen fiber bundles of rat reticular dermis are slim, loosely arranged. There are more morphological similarities between rat groups and human groups than pig groups.(2) Measurement results:There was no statistical difference of collagen fiber bundles’diameter between porcine reticular dermis and human’s (.P=0.34); collagen fiber bundles of rat reticular dermis is a little slender than human’s (P< 0.05). Gap rate measurements show that arrangement of collagen fiber bundles in porcine reticular dermis are more tight than human’s (P<0.01); but rat’s is similar to human’s (p=0.17).4. Picric acid and Sirius red staining:There are less type Ⅲ collagen in porcine dermis group, ratio of type Ⅰ and type Ⅲ collagen was significantly greater than the person group (P<0.05), but difference of these between rat and human group was not significant (P=0.72).5. Thermal analysis:In thermogravimetric analysis curves and derivative thermogravimetric curves, from 38℃ to 200 ℃, weight loss rate of human reticular dermis is biggest in three groups (11.58±0.80), followed by pig group (11.33± 2.02), the rate of the rat group minimum (10.07±2.83).Differential scan calorimetry curse indicate that porcine group is similar to human group, but rat group is different from human group.6. Fourier transform infrared spectroscopy scanning:The amide bands (e.g. Amide Ⅰ and Amide Ⅱ) observed in pig group showed broader band-widths than human one, but rat group is similar to human one. Moreover, pig and rat group showed more predominant amide A bands than the human one, especially rat one.Conclusions:Collagen fiber bundle’s diameter of porcine reticular dermis and their thermal analysis is similar to the human’s, moreover porcine reticular dermis has a larger area than rat ones, so it will have good prospects in preparation of larger area of xenogenic acellular dermal matrix. Collagen fiber bundles in rat dorsal reticular dermis arrange likely to human ones, and type Ⅰ and Ⅲ collagen content and their ratio is similar to the human’s, moreover their thermal stability is good, so rat dorsal reticular dermis will has excellent prospects in preparation of smaller areas of xenogenic acellular dermal matrix and even it’s clinical effects may be better than pig’s.