OBJECTIVETo investigate the role of fibroblasts in reconstruction of composite skin, and evaluate the effect of composite skin on full-thickness skin defect.

METHODSKeratinocytes and fibroblasts were seeded on the surface of acellular dermal matrix and cultivated in vitro to reconstruct the composite skin. Adherence of keratinocytes to dermal matrix was observed. Then take rate and histological construction were investigated after the composite skin was used to cover full-thickness skin defect wound in nude mice (n = 16).

RESULTSKeratinocytes grew and proliferated to reach tho confluence on the surface of the acellular dermal matrix. Keratinocytes adhered more stablely and could not be torn down from dermal matrix in operation when few fibroblasts were seeded on the epidermal surface of the dermal matrix. After grafting, the composite skin closed the full-thickness wound in nude mouse. The total survival was achived in 10 mice (62.5%). The newly generated skin was with intact histological construction of base membrance containing laminin and type IV collagen.

CONCLUSIONComposite skin could close the full-thickness wound, and fibroblasts could improve adherence of keratinocytes to dermal matrix, which should benefit the survival of composite skin.

Animals ; Cells, Cultured ; Fibroblasts ; Keratinocytes ; Mice, Nude ; Skin ; Skin Transplantation

OBJECTIVETo acquire lots of cell to culture during the primary cell culture.

METHODWe take the split-thickness skin slide technique to acquire the dissociated fibroblast cell in two big-ear rats.

RESULTSThe cell number is above 10(6) from 1 cm x 2 cm split-thickness skin slide and the technique is simple, economic, effectve.

CONCLUSIONWe think this way is better than other methods, and should be adopted in the primary cell culture, especially in fibroblast transplantation by injection.

Animals ; Cell Culture Techniques ; methods ; Fibroblasts ; cytology ; transplantation ; Rabbits

The present study aimed to assess the molecular profiles of subepithelial connective tissue grafts (CTGs) obtained at different locations and depths in the human palate. Sixty-four CTGs belonging to anterior deep (AD), anterior superficial (AS), posterior deep (PD), and posterior superficial (PS) groups were subjected to RNA-Sequencing and their transcriptomes were analyzed computationally. Functional correlations characterizing the CTG groups were validated by cell biological experiments using primary human palatal fibroblasts (HPFs) extracted from the CTGs. A clearly more pronounced location-dependent than depth-dependent difference between the grafts, with a minimal number of genes (4) showing no dependence on the location, was revealed. Epithelial, endothelial, and monocytic cell migration was strongly (P < 0.001) potentiated by AD- and PS-HPFs. Moreover, significantly increased expression of genes encoding C-C and C-X-C motif chemokine ligands as well as significantly (P < 0.01) activated p38 signaling suggested immunomodulatory phenotype for AD- and PS-HPFs. Increased growth factor gene expression and significantly activated (P < 0.001) Erk and Akt signaling in HPFs originating from A-CTGs implied their involvement in cell survival, proliferation, and motility. Prominent collagen-rich expression profile contributing to high mechanical stability, increased osteogenesis-related gene expression, and strongly activated (P < 0.001) Smad1/5/8 signaling characterized HPFs originating from P-CTGs. The present data indicate that in humans, differences between palatal CTGs harvested from different locations and depths appear to be location- rather than depth-dependent. Our findings provide the basis for future personalization of the therapeutic strategy by selecting an optimal graft type depending on the clinical indications.
Humans ; Connective Tissue/transplantation* ; Palate ; Collagen ; Fibroblasts ; Signal Transduction

OBJECTIVETo observe dynamically the influence of the application of dermal template on the p53 gene expression and apoptosis during wound repairing in burn patients.

METHODSTwenty burn patients were enrolled in the study and were divided into experiment (E, n = 11) and control (C, n = 9) groups. The escharectomy wounds in patients with 3rd degree burn in E group were covered with dermal template overlain with thin split-thickness autograft, while those in C group were covered with thin split-thickness autograft only. Specimens were harvested from wounds of both groups at 1st, 2nd, 3rd, 4th and 5th post operative week (POW). The P53 expression and the apoptosis were assessed respectively by immunohistochemistry and by TUNEL kit. The change in cell number was observed after HE staining.

RESULTSThe P53 expression increased gradually along with the wound healing process from 1st to 4th POW, which was significantly higher than that in C group at 2nd, 3rd, and 4th POW (P < 0.05), and it reached the peak at 4th POW. Fibroblasts underwent apoptosis at 1st POW in E group, while apoptosis of the endothelial cells occurred mainly at 2nd and 3rd POW. There was obvious difference in the rate of apoptosis between the two groups in 3rd and 4th POW (P < 0.05). The numbers of fibroblasts and vascular endothelial cells in E group were smaller than those in C group.

CONCLUSIONApplication of dermal template overlain with thin split-thickness autograft to wounds could induce P53 expression and cell apoptosis, thereby reduce scar formation, resulting in improvement of the quality of wound healing.

Adult ; Apoptosis ; Burns ; metabolism ; pathology ; therapy ; Dermis ; transplantation ; Fibroblasts ; pathology ; Humans ; Skin Transplantation ; methods ; Transplantation, Autologous ; Transplantation, Heterologous ; Tumor Suppressor Protein p53 ; genetics ; Wound Healing ; Young Adult

OBJECTIVETo evaluate the effects of mixed grafting of vascular endothelial cells and fibroblasts on the angiogenesis of artificial dermis.

METHODSFull-thickness skin defects with the size of 2. 5 cm x 2. 5 cm were made on the back of 27 Wistar rats, and they were randomized into endothelium group ( cultured vascular endothelial cells were mixed into 0. 5 ml fibrin glue, and evenly sprayed onto wound bed in density of 1.0 x 10(5)/cm(2) before artificial dermis grafting) , mixed grafting group( cultured vascular endothelial cells and fibroblasts were mixed into 0. 5ml fibrin glue, and evenly sprayed onto wound bed in density of 1.0 x 10(5)/cm(2) before artificial dermis grafting) , and control group( with spraying of equal amount of fibrin glue on the wound bed). Each group consisted of 9 rats and 18 wounds. On 5 and 10 post-grafting day (PGF) , the grafts and surrounding tissue were harvested for the observation of neo-vascular growth with HE staining, VEGF antibody staining, Masson's trichrome staining and India ink staining. Evans blue perfusion method was also used to measure the angiogenesis of micro-vascular vessels on 5 PGD.

RESULTSThe growth of new vessels was observed in each group on 5 PGD with HE staining, VEGF antibody staining, Masson's trichrome staining and India ink staining. Under high power field, the number of new vessels in endothelium and mixed grafting groups was (14. 2 3.6) and (12.1 +/- 2. 5) , which were obviously higher than that in control group (3.9 +/- 1.6, P < 0.05). On 10 PGD, angiogenesis and increased synthesis of collagen tissue were observed in both wound bed and artificial dermis. On 5 PGD, the amount of absorbed Even blue extracted from the grafted dermis in endothelium group (0. 167 +/-0. 058) and mixed grafting group (0. 155 +/- 0. 046) was significantly higher than that in control group (0. 066+/-0. 024, P <0. 05).

CONCLUSIONMixed grafting of homogenous vascular endothelial cells and fibroblasts is beneficial for the promotion of neovascularization, acceleration of angiogenesis and maturation of artificial dermis during the wound healing process.

Animals ; Cell Transplantation ; Dermis ; Endothelial Cells ; transplantation ; Fibroblasts ; transplantation ; Male ; Neovascularization, Physiologic ; Rats ; Rats, Wistar ; Skin ; blood supply ; Skin Transplantation ; Skin, Artificial ; Wound Healing

OBJECTIVETo explore a feasible method to repair full-thickness skin defects with tissue engineered techniques.

METHODSThe skin specimens were cut from the Changfeng hybrid swines' abdomen, then keratinocytes and fibroblasts were isolated and harvested by trypsin, EDTA and type II collagenase. The cells were seeded in petri dishes for primary culture. When the cells were in logarithmic growth phase, they were treated with dispase II (keratinocytes) or trypsin (fibroblasts) to separate them from the floor of the tissue culture dishes. A biodegradable material-pluronic F-127 was prefabricated and mixed with these cells, and then the cells-pluronic compounds were seeded evenly into polyglycolic acid (PGA). Tinally the constructs were replanted to autologous animals to repair full-thickness skin defects. Histological changes were observed in 1, 2, 4 and 8 weeks postsurgery.

RESULTSThe cells-pluronic F-127-PGA compounds could repair autologous full-thickness skin defects. Histologically, the tissue engineered skin was similar to normal skin with stratified epidermis overlying a moderately thick collageneous dermis.

CONCLUSIONTissue engineered skin can repair autologous full-thickness skin defects with primary-cultured keratinocytes and fibroblasts as seed cells and PGA as a cell carrier.

Animals ; Female ; Fibroblasts ; physiology ; Male ; Polyglycolic Acid ; pharmacology ; Skin Transplantation ; Skin, Artificial ; Swine ; Tissue Engineering

PURPOSE: Whole liver transplantation has limitation including donor shortage and fatal surgical complications. Hepatocyte transplantation, which is simpler and less expensive than whole liver transplantation, allows the use of living related donors, permits the use of a single donor organ for multiple recipients. However, hepatocytes have limitation in proliferation and lose their property during culture period. To over come these problems, here we performed differentiation of human embryonic stem cells (hESCs) into definitive endoderm in order to differentiate into hepatocytes efficiently. METHODS: Undifferentiated hESCs were maintained on mouse embryo fibroblast feeder (MEF) layer for 5~7 days. For endoderm differentiation, we used modified Kevin A D'Amour's method that added 100 ng/mL Activin A for 5 days. After differentiation, differentiated endodermal cells were collected and RT-PCR and immunostain analysis were performed. RESULTS: After 5 days of differentiation period, hES cells showed endoderm committed-cells and increased expression of endoderm-specific marker genes (Sox17 and Foxa2). Also differentiated endoderm cells were stained with Sox17 and Foxa2 whereas undifferentiated hES cells were not stained with Sox17, Foxa2. CONCLUSION: In vitro differentiotion from hES cells to definitive endoderm was done repetitively by our methods. Further well defined protocol for differentiation of definitive endoderm to hepatocytes should be made.
Activins ; Animals ; Embryonic Stem Cells* ; Embryonic Structures ; Endoderm* ; Fibroblasts ; Hepatocytes ; Humans* ; Liver Transplantation ; Mice ; Tissue Donors

PURPOSE: We attempt to heal the defect of meniscus using fibrin glue and allogenic meniscal cells in rabbit. MATERIALS AND METHODS: Meniscal cells were isolated from medial meniscus of knee joint of rabbit and cultured and expanded in vitro. The mixture of fibrin glue and meniscal cells implanted to the defect of rabbit meniscus. Examination of gross morphology, histological and immunohistochemical staining of retrieved specimens were performed at 3 and 6 weeks. RESULTS: Cultured cells uniformly displayed an elongated, fibroblastic and fibrocartilagenous morphology. The histological and immunohistochemical analysis revealed regeneration of meniscal tissue at the defect site. CONCLUSION: Meniscal cell transplantation with fibrin glue resulted in the regeneration of meniscus tissue successfully in rabbit model. This approach could be applied for treatment of meniscus injury.
Cell Transplantation ; Cells, Cultured ; Fibrin Tissue Adhesive* ; Fibrin* ; Fibroblasts ; Knee Joint ; Menisci, Tibial ; Regeneration ; Transplants

Animals ; Cellular Reprogramming ; Dentate Gyrus ; cytology ; Fibroblasts ; cytology ; Mice ; Neural Stem Cells ; cytology ; transplantation ; Swine

Cyclosporin A-induced gingival hyperplasia is frequently found in the patients who have been received an immunosuppressant for the organ transplantation. However, its exact mechanism is still unknown. The expression of FGF-5 and FGF-7 were studied in cyclosporine A-induced gingival hyperplasia (CGH) and inflammatory gingival hyperplasia (IGH). Immunohistochemistry and in situ hybridization were used for localization of protein and mRNA. The expression of FGF-5 and FGF-7 was different from CGH and IGH. FGF-5 and FGF-7 was strongly expressed in fibroblast in CGH (P<0.005 and P<0.05, respectively). FGF-5 mRNA was localized in the middle portion of connective tissue. FGF-7 mRNA was also identified in fibroblasts and mast cells. In conclusion, FGF-5 and FGF-7 were produced excessively by fibroblasts in CGH. Considering their known functions, their expression in CGH is important for production of collagen and proliferation of fibroblasts.
Collagen ; Connective Tissue ; Cyclosporine* ; Fibroblasts ; Gingival Hyperplasia* ; Humans ; Immunohistochemistry ; In Situ Hybridization ; Mast Cells ; Organ Transplantation ; RNA, Messenger ; Transplants