| Background and purpose:Mature teratoma come from the two or three germ layers of the adult-type tissue. Contents of mature cystic teratoma with ectodermal major organizations, including the skin, sebaceous glands, sweat glands, hair, some with teeth and nerve tissue. Teratoma contains skin structure,we call it Skin in Mature teratoma(SMT), can be used to repair the damage of the skin, if can be used for damage repair, can formation the skin with normal skin structure? The healing epidermis is the original damage the skin or teratoma tissues, which is what we observed teratoma raised new problems. And which is an important problem to be solved. At the same time, the skin in order to meet the clinical demand for skin grafts, long-term preservation of the mature teratoma skin equivalent is one of important issue. At present, the cryopreservation is the currently accepted the most effective method fore allogeneic skin grafts long-term preservation. There is no literature reported about and no experience on cryopreservation of mature teratoma skin equivalent.Materials and Methods:First, Human mature cystic teratoma tissue diagnosis and identification of SMT: Mature cystic teratoma tumor of the skin from ovarian mature cystic teratoma patients of Nanfang Hospital the Obstetrics and Gynecology department. Resected specimens of patients with ovarian mature cystic teratoma, Cut with a sterile surgical instruments for general observation of tumor, and take mature cystic teratoma with scalp hair, fixed with 10% neutral formaldehyde, dehydration, transparent, wax dipped, embedded, sliced and hematoxylin-eosin staining.Second, Results of SMT and BABL/c-nu/nu mouse skin morphological structure and immunohistochemical phenotypeHealth, SPF level BALB/c-nu/nu mice,4 to 8 weeks of age, the quality of 16-22g, purchased from Experimental Animal Center of Southern Medical University, all experimental mice were used in the Experimental Animal Center of Southern Medical University barrier Animal room feeding, temperature control at 28?. SMT and BABL/c-nu/nu mouse skin fixed in 10% neutral formaldehyde solution and paraffin embedded sections, HE staining, panCK (AE1/AE3), CK5/6, CK7, CK8, CK14, CD31, CD34, and ? immunohistochemistry were carried out.Thirdly,Human SMT xenograft to BABL/c-nu/nu mouse and identification: Healthy, clean-level BALB/c-nu/nu mice,4-8 weeks of age, the quality of 16-22g, purchased from Experimental Animal Center of Southern Medical University, all experimental mice were used in the Experimental Animal Center of Southern Medical University barrier Animal room feeding, temperature control at 28?. Before the transplant surgery, using sterile surgical instruments to collect mature teratoma skin equivalent cut into 1cm of skin tissue, remove subcutaneous adipose tissue and the surface hair, became full-thickness skin graft, then put into RPMI1640 medium for use.21 BABL/c-nu/nu mice were divided into experimental group (n=16) and control group (n=5). Experimental group,16 SMT planting to mice were from 13 specimens of patients. Animals were anesthetized using an intraperitoneal injection of pentobarbital sodium (40 mg/kg of body weight) and were placed in a prone position on a standard surgical platform with the dorsum exposed. Skin was disinfectioned and a square graft bed was created by excising approximately 1cm2 of full-thickness skin and subcutaneous tissue from the subscapular region of the mouse. In experimental group, the SMT placed in the graft bed, after fitting interrupted suture with 7-0 nylon line, directly over Vaseline gauze, glue Band-Aid. Control group, remove the skin and subcutaneous fatty tissue, made of about 1cm2 of skin defects, direct coverage of Vaseline gauze, glue Band-Aid. Continuous observation and general radiography. From the first to fourth weeks, mice were killed weekly, and the remaining 12 mice after 60 days were killed using 1% sodium pentobarbital anesthesia (80mg/Kg), the control group anesthesia to death mice using 1% pentobarbital sodium after 30 days. Remove the healing skin grafts and wound healing area tissue in the control group, fixed in 10% formalin, embedded in paraffin, routine histological analysis and immuno-histochemistry.Fourthly, Cryopreservation of SMT:Freezing:Obtain sterile teratoma skin equivalent made of the size of 1 cm2 to remove subcutaneous fat and some dermal tissue, put teratoma skin equivalent into vials, adding 1ml of the frozen buffer at 4? refrigerator for 30 minutes penetration; removal of vials of frozen buffer solution, add 1ml of the freezing liquid penetration in the refrigerator at 4? for 30 minutes;-20? refrigerator 30 minutes;-80? overnight (at least 12 hours);-198? liquid nitrogen.Thawing:The frozen pipe containing teratoma skin equivalent and frozen buffer solution removed from the liquid nitrogen quickly put into 37? water bath gently shake until the ice melted, frozen liquid with a pipette aspiration to join the recovery buffer,0.5 mol/L trehalose at room temperature for 15 minutes, then put teratoma skin equivalent into 6-well plate, each hole by adding 5ml of RPMI1640 medium. Then put plate into 5% CO2 37? incubator for 3 hours.After thawing, part of the teratoma skin equivalents underwent HE staining for morphological detection, Ki-67 immunohistochemistry and stereological morphological detection of frozen skin equivalents proliferation; part of the sample fixed with glutaraldehyde, transmission electron microscopy detected ultrastructural changes; part of the samples underwent tissue MTT assay to detect the biological activity of teratoma skin equivalent.V. Human SMT after cryopreservation xenograft to BABL/c-nu/nu mouse: Healthy, clean-level BALB/c-nu/nu mice,4-8 weeks of age, the quality of 16-22g, purchased from Experimental Animal Center of Southern Medical University, all experimental mice were used in the Experimental Animal Center of Southern Medical University barrier Animal room feeding, temperature control at 28?. Before the transplant surgery, using sterile surgical instruments to collect mature teratoma skin equivalent after cryopreservation cut into 1cm of skin tissue, remove subcutaneous adipose tissue and the surface hair, became full-thickness skin graft, then put into RPMI1640 medium for use. Intraperitoneal injection of 1% solution of sodium pentobarbital (40mg/kg) anesthesia in nude mice. Skin disinfection, the use of surgical instruments made of 1 cm skin wounds in the nude side of the back. Put teratoma skin equivalent after cryopreservation on the skin wounds, with dermis side down, interrupted suturing, with Vaseline gauze wrap, wrap Band-Aid, single cages and make a mark. After transplantation, observe the general condition and appearance of grafts, for four weeks, weekly cut graft and fixed in 10% neutral formaldehyde, routine HE staining were observed.2 months after transplantation, remove the graft fixation, HE staining were observed, CK5/6 immunohistochemical staining was used to identify origin of healing skin.?. SMT xenograft experiments impact on organs of BABL/c-nu/nu mouse:2 months after transplantation, experimental terminated with mice were killed using 1% sodium pentobarbital anesthesia, organs of mice fixied with 10% neutral formaldehyde, dehydrated, transparent, wax dipped, embedded, sliced and hematoxylin-eosin staining to detect changes in pathology.Results:First, Human mature cystic teratoma tissue diagnosis and identification of SMT: Teratoma patients with surgical specimens, initial screening the study required samples by general observation, according to whether the contents were skin equivalent with hair growth. The skin equivalent with hair growth removed, routine HE. staining, observed under the microscope, see the skin-like tissue covered the surface keratinized stratified squamous epithelium, sebaceous glands, dermis, sweat glands, hair, collagen fibers and nerves, blood vessels, which were specimens the study required.Second, Results of SMT and BABL/c-nu/nu mouse skin morphological structure and immunohistochemical phenotype:SMT epidermis was keratinized stratified squamous epithelium, the epithelium was thick, about 5-6 layers; sebaceous glands and hair follicles of SMT were bulky, hair follicles were fewer. mouse skin was thin, the epidermis was about 2-3 layers, mouse skin sebaceous glands with small size, follicle quantity and size small.Immunohistochemistry results of SMT and mouse skin showed that:panCK (AE1/AE3) were expressed in both SMT and mouse skin; CK5/6 in the epidermis and sebaceous of SMT were positive expression, and mouse skin were negative; CK7, CK8 in both mouse skin and SMT were negative; CK14 in the epidermis and sebaceous of SMT and in the epidermis and hair follicles of mouse skin were positive expression; CDS 1 in capillaries of SMT were positive expression, a weak positive expression of mouse skin blood vessels; CD34 in the stroma and vascular of SMT were strong positive expression, in mouse skin interstitial were weak expression; VIII expression in SMT capillaries were positive, there was a slight expression of hair follicles in mouse skin. Therefore, the mainly difference of immunohistochemistry results between SMT and mouse skin was in the CK5/6, epidermis and hair follicles of SMT were positive, but negative for mouse skin.Thirdly, Human SMT xenograft to BABL/c-nu/nu mouse and identification: Teratoma skin equivalents transplanted into BABL/c-nu/nu mice,1 week after transplantation, the teratoma skin graft was milky white, the surrounding light yellow, soft texture; graft epithelial and sebaceous gland tissue degeneration, focal necrosis can be seen, but the graft connected with mouse fascia tightly, rich in capillaries seen in the epidermis.2 weeks after transplantation, graft-light yellow, brown around the yellow, soft texture; skin equivalent graft and surrounding skin of nude mice skin tight, the surrounding epithelial cell regeneration, and gradually to the central growth, the surface layer of keratosis and necrosis can be seen epithelium. Dermis sebaceous glands, sweat glands, and new hair.3 weeks after transplantation, graft surface to form thick crusts, brownish yellow, hard, after crusts scab off a milky white skin tissue, the surface scattered pores; epidermal keratinizing squamous epithelium, sebaceous glands, dermis, sweat glands, hair and mature adipose tissue.4 weeks after transplantation, part of the graft can be seen pigmentation, were brown, visible keratosis epidermis and Skin appendages, and visible hair growth. Appearance and function of graft repair to be 3-4 weeks,25-35 days or so visible surface of the skin to grow new hair.In the control group, there was scar formation and contraction in the damaged skin area of BABL/c-nu/nu mice after 1 month operation, under microscope, Stratified epithelium lined on the body surface, collagen tissue proliferated in the dermal layers, no sebaceous glands and hair follicles were observed.Transmission electron microscopy will detect graft after transplantation, skin graft healing between the hemidesmosomes and basement membrane structure is clear, and the basement membrane hemidesmosomes of nude skin loose, the structure is not clear. Basal cell of graft melanocytes and melanin granules can be seen, but the nude mouse skin is thin compared teratoma skin equivalent graft, observe the layer structure of mouse skin, the melanin granules and melanin were not found in all cells present. It can explain the healing graft was not skinof nude mice, but teratoma skin equivalent.Before transplantation, cytokeratin(CK) immunohistochemical staining was performed on mouse skin and teratoma skin equivalent, CK7, CK8 in both mouse skin and teratoma skin equivalent epithelial cells were negative, AE1/AE3, CK14 were both expressed in mouse skin and teratoma skin equivalent, and the expression of CK5/6 in teraroma skin equivalent epithelial cells and sebaceous glands was strong positive, but in mouse skin epithelium was negative. Thus, CK5/6 as a distinction between mouse skin and teratoma skin equivalent epithelial markers. The graft and around mouse skin were cut and fixed, CK5/6 immunohistochemistry showed that expression of CK5/6 nude mice skin were negative, while there was visible CK5/6 expression in the graft epidermis and epithelial nests of dermis layer. Combined with the general observation after transplantation, we can confirm that the graft was teratoma origin. Employing vascular markers CD31,? marked skin graft healing dermal blood vessels, no vascular expression. So that, the healing of graft blood vessels to provide blood supply mainly from the mouse.Fourthly, Cryopreservation of SMT:Teratoma skin equivalent after cryopreservation maintain intact epidermis and dermis structure, epidermis and dermis was no degeneration and ice crystal formation, there was no separation between the epidermis and dermis. TEM results showed that the hemidesmosomes between epidermis and basement membrane structure was intact, and there was no damage in desmosomes between cells connected institutions, organelles in the cytoplasm such as mitochondria, ribosomes, tension wire structure did not change significantly from frozen skin tissue sections, endoplasmic reticulum was observed in cell cytoplasm. Ki-67 is positive in cell nuclei. Ki-67 expression of frozen teratoma skin equivalent under the microscope was intensity and the positive rate was similar to fresh. Moreover, PU value of Ki-67 protein determination was carried out on the fresh and cryopreserved skin equivalent. Ki-67 protein PU in the fresh teratoma skin equivalent is 26.098±1.202, Ki-67 protein PU in cryopreserved samples is 27.008±1.480 PU, Statistics showed that there was no significant difference between the fresh and cryopreserved group(P=0.816). The viability index of fresh skin equivalent was 0.090±0.017 (0.068-0.117) under MTT assay, the viability index of cryopreserved skin equivalent was 0.076±0.012 (0.062-0.097), percentage viabilty index of cryopreserved samples was 86.17±9.23%.?. Human SMT after cryopreservation xenograft to BABL/c-nu/nu mouse:After transplantation, the cryopreserved graft healed the skin well.2-4 weeks after transplantation, surface layer of the skin brown crusts fall off, the exposed tissue was white, the grafts closely connected with the surrounding mice skin, opening pores scattered spots.2 months after transplantation, healing graft was milky white, the pores scattered, no hair grow, healing graft was observed under the microscope, the surface is keratinized squamous epithelial, showing openings of hair follicles, the dermal layer see a small number of sweat glands, sebaceous glands and hair did not see. Cytokeratin5/6 immunohistochemistry staining method to identify the healing graft origin. The surface of graft no CK5/6 expression. sweat gland and epithelial nests in dermis was positive expression organizations. the expression of CK5/6 was positive in epidermis, sweat glands and sebaceous glands of teratoma skin equivalent before and after cryopreservation, cryopreservation had no effect on CK5/6 antigen expression. Therefore, some of the healing epidermis was the teratoma skin equivalent, the rest was mice skin. From the CK5/6 expression area, epithelial nests and sweat glands in dermis was teratoma skin equivalent, while the surface of the epidermis was the skin of nude mice.?. SMT xenograft experiments impact on organs of BABL/c-nu/nu mouse:Two months after grafting, generally observed organs of BABL/c-nu/nu mouse, heart, liver, spleen, lungs, kidneys and other organs had no obvious lesions. Pathology under the microscope to observe the change of organs. heart:Microscopically, myocardial fiber short column, and a branch. Vascular dilatation and congestion; liver:structural integrity of hepatic lobule, central vein and sinusoidal no dilatation and congestion, no liver cell necrosis; spleen:red pulp, white pulp, structural integrity, red pulp splenic sinus congestion; Lung:bronchial and alveolar structure integrity, interstitial vascular dilatation and congestion; brain, kidney, adrenal gland, pancreas, ovary, uterus, stomach and small intestine tissue structural integrity show no obvious lesions.Conclusion:This was the first time that we discusseed SMT xenograft problem, and following innovations was made:First, the skin-like structural components in mature teratoma can be recycled, can be used as a naive skin to repair the damaged skin and restoration effect is very satisfactory, as follows:the growth of healing grafts was in good condition, there were visible black hair growth. Microscopically, skin healed well, sebaceous and sweat glands dermis, the hair structure were be observed. Pigmentation appeared in healing graft.Second, SMT can be used for Cryopreservation, recovery and transplantation. After Deep-freezing, the morphology and ultrastructure structures of SMT was intact, even after their recovery, the cryopreserved SMT has the same proliferation as fresh SMT, and the vibility index percentage was 86.17%. After recovery, the deep-frozen SMT transplanted to BABL/c-nu/nu mice, the SMT healed well, the appearance was white, part of healing skin was SMT, and the part was BABL/c-nu/nu mice skin, both SMT and mouse skin participated in the repair of skin wounds.Thirdly, after SMT transplanted to the BABL/c-nu/nu mice, no substantial damage to organs, and no tumor formation.Fourthly, healing skin growed new hair, so we think the future of this technology can be used for the treatment of baldness. After freezing SMT can not only repair the damaged skin, and the appearance was white, and this appeared to resolve the problem of pigmentation after skin transplantation. After cryopreservation, no hair growth in healing skin, but the sweat gland function exists, it can meet the need of no hair growth site (such as the palm side) of the transplantation, and still preserved skin perspiration function.V. CK5/6 can be used for identification between SMT and BABL/c-nu/nu mouse skin, SMT epithelium was CK5/6 positive, BABL/c-nu/nu mouse skin was CK5/ 6 negative; In addition, CD34 has some value in identification between SMT and BABL/c-nu/nu mouse skin,SMT was interstitial CD34 positive,BABL/c-nu/nu skin of mice was CD34 weakly positive. |
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