OBJECTIVETo culture the keratinocytes on the acellular pig dermis and establish a composite skin in vitro.

METHODSFull thickness skin taken from neonatal SD rats of approximate 24-hour-old was cultivated in asepsis condition, which was then separated into epidermal and dermal layers with low-temperature enzyme digestion. The basal lamina cells between the two layers were scraped off and the pure keratinocytes were obtained using gradient density centrifugation. In the experimental group, the keratinocytes were cultured on acellular xenodermis(AX) as the three-dimensional frames. In the control, the keratinocytes were cultured without any trophoblast. The air-liquid interface(ALI) culture continued after the primary culture. Routine histological HE staining and assay of Pancytokeratin and Laminin with strepavidin-biotin-peroxidase complex (SABC) method were used to study the morphology of CK and AX.

RESULTSHE staining demonstrated the formation of more than 4 lays of keratinocytes and basement membrane, with slight keratinization of the cells. Pancytokeratin(+) in immunohistochemical results confirmed that the cultured cells on AX were keratinocytes, not other kinds of cells. Laminin(+) indicated that new procreant basement membrane appeared in CK.

CONCLUSIONKeratinocytes cultured on the acellular pig dermis grow well and form basement membrane. This study implies successful construction of composite skin.

Acellular Dermis ; Animals ; Animals, Newborn ; Basement Membrane ; cytology ; Cell Culture Techniques ; Cells, Cultured ; Epidermis ; cytology ; Keratinocytes ; cytology ; Keratins ; analysis ; Laminin ; analysis ; Rats ; Skin ; cytology ; Skin Transplantation ; Staining and Labeling ; methods ; Swine ; Transplantation, Heterologous

OBJECTIVETo study the effect of platelet-derived growth factor-BB (PDGF-BB) in different concentrations on proliferation of tendon cells cultured in vitro.

METHODSRat tendon cells were cultured and identified in vitro. The rat tendon cells were cultured in PDGF-BB nutrient solution in different concentrations. They were then divided into 1, 5, 10, 20, 50, 100, 150, 200, 250 ng/mL PDGF-BB groups (cultured with 0.1 mL 0.5% PBS with addition of 1, 5, 10, 20, 50, 100, 150, 200, 250 ng/mL PDGF-BB respectively). Tendon cells in control group were cultured with 0.1 mL 0.5% FBS. Proliferation of tendon cells was detected by MTT test. The absorbance values of tendon cells in control group and 20 ng/mL PDGF-BB group before culture and after cultured for 12, 24, 36, 48, 60, 72 hs were determined.

RESULTSThe isolated cells were identified to be rat tendon cells as they were Type I collagen staining positive and TypeIII collagen staining negative. Compared with that of control group, the absorbance values of other groups were all increased, except for that of 250 ng/mL PDGF-BB group (P < 0.05 or P < 0.01). Besides, the absorbance value rose gradually with the increase of the concentration of PDGF-BB on, and then diminished gradually with the increase of the concentration of PDGF-BB from 20 ng/mL on. Tendon cells in 20 ng/ml PDGF-BB group began to increase in number when cultured for 12 hs, and it reached the highest level (0.53 +/- 0.04) at 48 h, which were obviously higher than those of control group at 24 - 72 h (P < 0.01). The absorbance value of tendon cells in 20 ng/mL PDGF-BB group was significantly higher than that of control group at 24, 36, 48, 60, 72 h after culture (P < 0.01).

CONCLUSIONSPDGF-BB can promote the proliferation of tendon cells in a definite range of concentration and time.

Animals ; Cell Proliferation ; drug effects ; Cells, Cultured ; Culture Media ; Dose-Response Relationship, Drug ; Platelet-Derived Growth Factor ; administration & dosage ; pharmacology ; Proto-Oncogene Proteins c-sis ; Rats ; Rats, Sprague-Dawley ; Tendons ; cytology ; drug effects

OBJECTIVETo study the effect of PDGF on dermal blood vessel reconstruction by transplanted tissue-engineering skin containing PDGF-B gene to rats.

METHODSThe recombined eukaryotic expression vector, pcDNA3.1-hPDGF-B, was constructed and transfected into fibroblasts mediated by LipofectAMINE. Keratinocytes + acellular dermal matrix (group A), keratinocytes + acellular dermal matrix + fibroblasts (group B), keratinocytes + acellular dermal matrix + fibroblasts with PDGF gene (group C) were recombined respectively, then transplanted them to rat dorsum and evaluated the reconstruction of blood vessels in the dermis after 2, 4, 6 week postoperation.

RESULTSIn 2-4 weeks after skin grafting the vascularization rate in group C was higher than that of group B and group A. The vascularization rates in all groups had no significant differences in six weeks (P > 0.05).

CONCLUSIONPDGF-B gene plays an important role in reconstruction of blood vessels in the dermis at early tissue-engineering skin grafting, which ensures the take of grafted tissue-engineering skin.

Acellular Dermis ; Animals ; Male ; Neovascularization, Physiologic ; Proto-Oncogene Proteins c-sis ; genetics ; Rats ; Rats, Sprague-Dawley ; Skin ; blood supply ; Skin Transplantation ; Skin, Artificial ; Swine ; Tissue Engineering ; methods ; Transfection

OBJECTIVETo establish a new method for the preparation of porcine acellular dermal matrix.

METHODSThe antigenicity of the porcine dermis was weakened by removing epidermal and dermal cells from the porcine skin through the digestion with low-concentration trypsin and repeated freeze-thaw cycles. Split thickness porcine skin was treated with 0.05% trypsin to remove the cells from the epidermis and dermis. Repeated freeze-thaw cycles were employed to further weed out the residual cells within the dermis. The prepared acellular dermis was then examined grossly, as well as histologically, and also by immunohistochemical method.

RESULTSNo cell could be identified in the prepared porcine acellular dermal matrix. The integral basement membrane was preserved on the surface of dermal matrix with compact dermal matrix collagen structure.

CONCLUSIONLow concentration trypsinization and repeated freeze-thaw cycles seemed to be a simple and effective method for the preparation of xenogeneic acellular dermal matrix.

Animals ; Dermis ; cytology ; transplantation ; Extracellular Matrix ; transplantation ; ultrastructure ; Freezing ; Skin Transplantation ; Swine ; Tissue Engineering ; methods ; Trypsin ; administration & dosage

OBJECTIVETo investigate the effect of hepatitis B virus(HBV) X gene on the expression of SPG21.

METHODSThe expressions of SPG21 mRNA and protein in HepG2 and HepG2.2.15 cells were tested by RT-PCR and western blot. HepG2 cells were co-transfected with reporter plasmid pGL3-SPG21 and plasmids carrying individual genes of HBV, the luciferase activity was measured and the expressions of SPG21 were detected by RT-PCR and western blot.

RESULTSThe expressions of SPG21 mRNA and protein were higher in HepG2.2.15 cells than in HepG2 cells (0.36+/-0.06 vs 0.21+/-0.05, P value is less than 0.05). The activity of SPG21 in HepG2 cells transfected with pCMV-X was higher (875+/-27 vs 67+/-12, P value is less than 0.01) as compared to blank control group (transfected with pCMV-tag2B). HBV X gene enhanced SPG21 gene promoter activity, SPG21 mRNA expression and SPG21 protein production in HepG2 cells in a dose-dependent manner.

CONCLUSIONHBV X gene can specially activate SPG21 expression.

Adaptor Proteins, Signal Transducing ; genetics ; metabolism ; DNA, Viral ; genetics ; Hep G2 Cells ; Hepatitis B virus ; genetics ; Humans ; RNA, Messenger ; genetics ; Trans-Activators ; genetics ; Transfection

OBJECTIVETo investigate the aesthetic effect of wound repair with flaps.

METHODSOne thousand nine hundred and ninety-six patients with 2082 wounds hospitalized from January 2004 to December 2011. These wounds included 503 deep burn wounds, 268 pressure sores, 392 soft tissue defects caused by trauma, 479 soft tissue defects due to resection of skin cancer and mole removal, 314 soft tissue defects caused by scar excision, and 126 other wounds. Wound area ranged from 1.5 cm x 1.0 cm to 30.0 cm x 22.0 cm. Sliding flaps, expanded flaps, pedicle flaps, and free flaps were used to repair the wounds in accordance with the principle and timing of wound repair with flaps.

RESULTSFive flaps showed venous congestion within 48 hours post-operation, 2 flaps of them improved after local massage. One flap survived after local heparin wet packing and venous bloodletting. One flap survived after emergency surgical embolectomy and bridging with saphenous vein graft. One flap showed partial necrosis and healed after skin grafting. The other flaps survived well. One thousand three hundred and twenty-one patients were followed up for 3 months to 2 years, and flaps of them were satisfactory in shape, color, and elasticity, similar to that of normal skin. Some patients underwent scar revision later with good results.

CONCLUSIONSApplication of suitable flaps in wound repair will result in quick wound healing, good function recovery, and satisfactory aesthetic effect.

Adolescent ; Adult ; Aged ; Aged, 80 and over ; Burns ; surgery ; Child ; Child, Preschool ; Esthetics ; Female ; Humans ; Male ; Middle Aged ; Reconstructive Surgical Procedures ; methods ; Soft Tissue Injuries ; surgery ; Surgical Flaps ; Wound Healing ; Young Adult