Comparison Of Between Neonatal Bovine And Adult Human Skin Histological And Biomechanical Features And Preparation Of Bovine Acellular Dermal Matrix

Background: The key treatment for extensive burns or trauma patients is how totimely and effective closure the wound. Lack of dermis is the main factor that effects theskin graft surgery in patients. So how to improve the prognosis of burns, trauma, andorthopedic patients and improve patient’s quality of life is very important. The dermistransplantation and/or dermal studies is one of the key to resovle the problem. Biologicalscaffolds composed of extracellular matrix (ECM) has been applied to a variety of surgicalreconstruction, and it was widely used as a strategy in tissue and organ replacement andregenerative medicine. Those biologic scaffold materials composed of ECM are harvestedfrom several different species. The ECM based biomaterials have significant mechanicalstrength, retained biological activity and can promote the regeneration of tissue. So thoseprotein-based materials have many inherent advantages over synthetic polymer materialsfor surgical and regenerative medicine applications. Allogeneic dermal source is limited andexpensive, so it is necessary to choose the heterogeneous dermal sources which arerelatively close to a human dermal. On the basis of the preliminary work of our group, wehave chosen a newborn bovine skin to observe the organizational structure andbiomechanical performance characteristics in comparison with human skin, provide theexperimental basis for the application of bovine dermal matrix. We explored the method ofpreparation of neonatal bovine acellular dermal matix. We used osmotic shock combinedwith sonication and detergent treatment methods produced ideal acellular dermal matrix,and evaluated the acellular dermal matrix histological and biological active ingredients innative dermal matrix.Objectives1. Through the comparative study of the morphological and biomechanical performances between newborn bovine and adult skin tissue, looking for similarities anddifferences between the two aspects, finding the evidences for the clinical application ofbovine dermal matrix.2. To explore the preparation of newborn bovine acellular dermal matrix, and observethe changes of the dermal matrix histology and biological active ingredients before andafter treatment.Methods1. The dorsal full-thickness of samples were stained by hematoxylin and eosin (HE)staining, Masson’s trichrome staining, sirius redstaining and Gomori staining, and wereexamined under light microscopy. These samples were also observed by scanning electronmicroscopy (SEM) and transmission electron microscopy (TEM). The relevant data ofhistological images were measured with image analysis software. Those samples weredetected material testing machine for biomechanical properties.2. Collection and preparation of the newborn bovine reticular layer of dermis.Respectively, using the enzymatic method, osmotic shock combined with sonication anddetergent method to explore the method of prepared acellular dermal matrix. And thenusing gross observation and HE staining to evaluate the preparation method.3. The prepared acellular dermal matrix carried out HE staining, DAPI staining andnucleic acid electrophoresis to detect residues cell composition. The structure of collagenbundles and cell composition were observed by scanning electron microscope (SEM);PicoGreen method, DMMB method and the BCA method analysis the content of DNA,sGAG and protein before and after the acellular dermal matrix, respectively. ELISA methodwas used for the determination of TGF-β1and EGF, bFGF and KGF contents the acellulardermal matrix before and after treament.Results1. The newborn bovine skin compared with human skin, the thickness of the dermisand epidermis were significantly thinner, the ratio between the dermis and epidermis wassignificantly reduced. The gap rate between bundles [(41.72±1.81) vs (40.66±1.40)]and the caliber of the bundles [(11.28±0.18) vs (10.88±0.66)] were not significant instatistics (P=0.467，P=0.368).2. From the observation of scanning electron microscopy (SEM) examination, the dermal collagen bundles of newborn bovine were thinner and rarefaction arranged.Compared with human dermis, the newborn bovine had longer cyclical transverse striation,and the collagenous fibrils had no significant difference by transmission electronmicroscope (TEM).3. The maximum stress of newborn bovine (21.08±0.91) MPa were stronger than thatof human dermis (12.76±1.60) MPa (P=0.001), the elastic modulus were also strongerthan that of human [(82.12±1.23) MPa vs (48.63±5.50) MPa (P=0.00)]. The maximumstrain was much lower than that in human dermal (0.51±0.002) mm vs (0.75±0.028) mm(P=0.001).4. Enzymatic method can be prepared for acellular dermis, but it required the uniformthickness and the special parts of the reticular layer dermis. From the HE stainingobservation, the nuclei was removed using osmotic shock combined detergent methodprepared acellular dermis, but the use of detergent has a certain extent destroyed to collagen,the realization of the collagen fibers of the loose part of the lumps. Morphologicalobservation on decellularized dermis revealed the integral three-dimensional structure,interspersed collagen bundles, satisfying the removal of cells and their debris using osmoticshock combined with sonication and detergent treatment prepared acellualr dermis, andDAPI staining demostrate no DNA coloring in the dermis.5. Scanning electron microscopy results showed that the collagen fiber bundlesarranged in loose, no cell debris was found. The composition quantitative analysisconfirmed that the DNA content [(2516.1±324.2) ng/mg vs (249.5±53.8) ng/mg, P=0.000)], decreased by90.1%, sGAG content [(5.92±0.50) μg/mg vs.(2.48±0.24) μg/mg, P=0.000], retained about41.9%. Acellular, water content declined (72.83+1.43)%vs(69.68+1.78)%, P <0.05]. Cytokines in TGF-β1content (478.8±196.1) pg/g vs (180.3±111.0) pg/g, P=0.009], and EGF content (10.52±2.78) pg/g vs failed to detect the EGFcontent] were significantly reduced; bFGF content [(788.6±333.8) pg/g vs (364.8±294.8)pg/g, P=0.424] and a decrease of53.7%, KGF content [(0.033±0.00019) pg/g vs(0.033±0.00022) pg/g, P=0.433] was not changed significantly.Conclusion1. The dermis of newborn bovine and human are similar in three dimensional structureof collagen fiber. Although there have a difference in biomechanical properties, the decellularization dermal matrix can used as biomaterials for reparing the human dermis.2. Osmotic shock combined with sonication and detergent treatment could effectivelyachieve acellular extracellular matrix with integral histological structure and partiallypreserved major bioactive components of the newborn bovine dermis. These results providecritical experimental evidences for further improving the preparation of acellular dermis.