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

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

OBJECTIVETo construct of tissue engineering skin including active composite dermal matrix.

METHODSThe human fibroblasts and bovine collagen with type I were inoculated on the surface of porcine acellular dermal matrix (PADM) for construction of active dermal substitute, then epidermal cells were inoculated on the dermal matrix for gas-liquid interface culture. The tissue-engineering skin was observed by histological examinations.

RESULTSThe structure of fibroblasts in collagen was intact, which was used to construct composite dermal matrix with PADM. The epithelial structure of tissue-engineering skin was similar to that of normal skin with good cell differentiation. Some phenomena were showed in epidermis: basic layer, stratum spinosum, granular layer and stratum corneum, desmosomes.

CONCLUSIONFibroblasts-Collagen-PADM can be an optimal dermal matrix for construction of tissue-engineering skin.

Animals ; Cattle ; Cell Culture Techniques ; Collagen Type I ; Dermis ; transplantation ; Epidermis ; cytology ; Extracellular Matrix ; transplantation ; Fibroblasts ; cytology ; Humans ; Skin ; cytology ; Skin, Artificial ; Swine ; Tissue Engineering

To prepare scaffolds for heart valve tissue engineering, porcine heart valves were treated with varied concentrations of trypsin for 32, 56, 80 and 104 h or followed with DNase. And then the structure of acellular valves was observed under light microscope, scanning and transmission electron microscope. Porcine endothelial cells, human endothelial cells, and canine myofibroblasts were reseeded onto the acellularized porcine heart valve scaffolds once a day for 3 days. The valves were analyzed by immunohistochemical staining and electron microscopy. Results show that all endothelial cells and the majority of interstitial cells were removed from the heart valves after digestion with trypsin for 104 h, and the collagen fiber structure remains intact, but the space between collagen fibers increased slightly. Incubation with trypsin for 80 h and then with DNase almost removed all cells, and the collagen fiber structure and the space between the fibers remain intact. After reseeding, human endothelial cells almost fully cover the valve scaffold surface as shown by H-E staining and platelet endothelial cell adhesion molecules (PECAM-1) staining. Xenogeneic porcine endothelial cells also adhered to and grew on the scaffolds. As shown by H-E staining and actin staining, canine myofibroblasts not only adhered to the surface of valve scaffold but also migrated to the inner part of matrix after one week culture. These results suggest that the digestion of porcine heart valves with trypsin combining with DNase is a suitable method to remove cells. The acellular porcine heart valve scaffolds have a quite favorable biocompatibility with human and porcine endothelial cells as well as canine myofibroblasts.
Animals ; Bioprosthesis ; Cells, Cultured ; Endothelium, Vascular ; cytology ; transplantation ; Fibroblasts ; cytology ; Heart Valve Prosthesis ; Heart Valves ; cytology ; Humans ; Muscle Fibers, Skeletal ; cytology ; Swine ; Tissue Engineering

Our objective is to determine the quality of tissue engineered human skin via immunostaining, RT-PCR and electron microscopy (SEM and TEM). Culture-expanded human keratinocytes and fibroblasts were used to construct bilayer tissue-engineered skin. The in vitro skin construct was cultured for 5 days and implanted on the dorsum of athymic mice for 30 days. Immunostaining of the in vivo skin construct appeared positive for monoclonal mouse anti-human cytokeratin, anti-human involucrin and anti-human collagen type I. RT-PCR analysis revealed loss of the expression for keratin type 1, 10 and 5 and re-expression of keratin type 14, the marker for basal keratinocytes cells in normal skin. SEM showed fibroblasts proliferating in the 5 days in vitro skin. TEM of the in vivo skin construct showed an active fibrocyte cell secreting dense collagen fibrils. We have successfully constructed bilayer tissue engineered human skin that has similar features to normal human skin.
Fibroblasts/*cytology ; Keratinocytes/*cytology ; Mice, Nude ; Microscopy, Electron ; Microscopy, Electron, Scanning ; Quality Control ; Regeneration/physiology ; Skin/pathology ; Skin Transplantation/pathology ; Skin Transplantation/*standards ; Tissue Engineering/*standards

In covering wounds, efforts should include utilization of the safest and least invasive methods with goals of achieving optimal functional and cosmetic outcome. The recent development of advanced wound healing technology has triggered the use of cells to improve wound healing conditions. The purpose of this review is to provide information on clinically available cell-based treatment options for healing of acute and chronic wounds. Compared with a variety of conventional methods, such as skin grafts and local flaps, the cell therapy technique is simple, less time-consuming, and reduces the surgical burden for patients in the repair of acute wounds. Cell therapy has also been developed for chronic wound healing. By transplanting cells with an excellent wound healing capacity profile to chronic wounds, in which wound healing cannot be achieved successfully, attempts are made to convert the wound bed into the environment where maximum wound healing can be achieved. Fibroblasts, keratinocytes, adipose-derived stromal vascular fraction cells, bone marrow stem cells, and platelets have been used for wound healing in clinical practice. Some formulations are commercially available. To establish the cell therapy as a standard treatment, however, further research is needed.
Blood Platelets/metabolism ; Cell- and Tissue-Based Therapy ; Diabetes Mellitus, Type 2/complications/pathology ; Fibroblasts/cytology/transplantation ; Humans ; Keratinocytes/cytology/transplantation ; Stromal Cells/cytology/transplantation ; Tissue Engineering ; Ulcer/etiology/therapy ; *Wound Healing

Patient own fibrin may act as the safest, cheapest and immediate available biodegradable scaffold material in clinical 1 tissue engineering. This study investigated the feasibility of using patient own fibrin isolated from whole blood to construct a new human cartilage, skin and bone. Constructed in vitro tissues were implanted on the dorsal part of the nude mice for in vivo maturation. After 8 weeks of implantation, the engineered tissues were removed for histological analysis. Our results demonstrated autologous fibrin has great potential as clinical scaffold material to construct various human tissues.
*Biocompatible Materials ; *Bone Transplantation ; Cartilage/*transplantation ; Cell Division/physiology ; Culture Media ; *Fibrin ; Fibroblasts/cytology ; Mesenchymal Stem Cells/cytology ; Mice, Nude ; Organ Culture Techniques ; Periosteum/cytology ; *Skin Transplantation ; *Tissue Engineering

OBJECTIVETo investigate the influence of vascular endothelial growth factor (VEGF) gene modification on skin substitute grafted on nude mice.

METHODSHuman fibroblasts were transfected with VEGF adenovirus vector. Then the genetic modified fibroblasts were seeded on patches of Integra artificial skin. Twenty-four hours later, the Integra patches were grafted onto full-thickness skin defects on nude mice. Seventy-two nude mice were divided into experiment (n = 18, E, with fibroblasts seeded on Integra which were transfected by adenovirus containing VEGF in advance), GFP control (n = 18, the fibroblasts were transfected with adenovirus containing labelled GFP segment as same as that in E group, but containing no VEGF gene), Fb control (n = 18, without gene transfection), and control (n = 18, no fibroblast was seeded on Integra) groups. The survival rate, the revascularization process and the histological changes in the grafts in gene modified group (experimental group) and control groups were observed and analyzed.

RESULTSThe revascularization condition in the experimental group was much better than that in the control group. The grafts adhered firmly to the wound during early postoperation stage, and were more prone to bleed when separated from the wound. The survival rate was obviously higher, while the infection rate was much lower in experimental group (100.0%) compared with the control groups (83.3%, 75.0%, 77.8%, respectively) (P < 0.05).

CONCLUSIONHigh expression of VEGF by gene modification can promote the vascularization process of skin substitute, hence improve the grafting result.

Amino Acid Motifs ; Animals ; Cells, Cultured ; Female ; Fibroblasts ; cytology ; Humans ; Mice ; Mice, Nude ; Neovascularization, Physiologic ; genetics ; Skin ; cytology ; Skin Transplantation ; Skin, Artificial ; Transfection ; Vascular Endothelial Growth Factor A ; genetics

OBJECTIVETo observe the effect and safety of autologous cultured skin fibroblasts transplantation for treating depressed facial skin defects.

METHODSA total of 19 patients were treated from Jan, 2010 to Oct, 2010. Autologous skin fibroblasts were separated from postauricular skin biopsy or resected skin tissue in other surgeries such as blepharoplasty. They were cultured and expanded with exclusive method. Cells (2 x 10(7)/ml) within three passages were injected intradermally at the site of skin depression three times at one-month interval. Adverse events were observed and recorded. Clinical effects were evaluated and graded by two unrelated physicians before and 6 months after the first injection.

RESULTSCells from 16 patients were successfully cultured at the first time. The other 3 patients underwent a second harvest. A total amount of 6 x 10(8) cells could be reached within three passages in 45 days. 16 out of 19 patients accomplished the whole course of this study. Minor adverse events were observed in two patients including small ulcer caused by over injection in one patient and slightly redness and swelling in the other. The redness disappeared after a week without any treatment. No serious complications were observed. Significant difference was noticed between the scores obtained before and after the treatment.

CONCLUSIONSFrom this study, neither serious complications nor excessive cell proliferation or scar formation was found after cell injection. The effect of using autologous fibroblast transplantation was obvious and long-lasting, which provides a new choice for the treatment of depressed facial skin defects.

Adult ; Cells, Cultured ; Cicatrix ; therapy ; Face ; abnormalities ; Female ; Fibroblasts ; transplantation ; Humans ; Male ; Middle Aged ; Skin ; cytology ; Transplantation, Autologous ; Treatment Outcome ; Young Adult

OBJECTIVETo study the effect of tissue-engineered skin loaded with keratinocyte growth factor (KGF) nanocapsules for skin defect on athymic mice.

METHODSThe acellular dermal matrix (ADM) loaded with KGF-ADM was constructed by means of phacoemulsification solvent evaporation and low temperature drying. The human epidermal stem cells and fibroblasts were captured and identified, then cultivated on the surface of the KGF-ADM. The cell growth was observed. The tissue-engineered skin without KGF was used as sham group. The autogenous skin graft was used as control group. 2 and 6 weeks after the skin was transplanted to the back of athymic mice, the contraction and histological healing of the transplanted skins were observed respectively. Then the immunofluorescence examination with anti-human K10-FITC and beta1-integrin-Cy3 were applied to detect the origin, growth and differentiation of epidermal and dermal cells in tissue-engineered skin.

RESULTSThe epidermal stem cells grew well and attached tightly on KGF-ADM. There were small round stem cells and polygonal terminally-differentiated cells, which appeared a partly cloning growth and a tendency of merging. The tissue-engineered skin with KGF nanocapsules gained better result in repairing the skin defects as compared with the blank group and the control group 2 and 6 weeks after transplantation. The regenerative skin cells could connect and mix closely with the athymic mouse skin cells on the border of skin defect. Meanwhile, the regenerative skin existed some contraction. The histological observation with HE staining showed that the regenerative skin possessed intact epidermis with several cell layers and normal keratose stratum, among which there were still some beta1-integrin (+) cells which represented epidermal stem cells or transient amplifying cells when they were tested by immunofluorescence after 6 weeks of transplantation.

CONCLUSIONSThe tissue-engineered skin loaded with KGF nanocapsules had a better result in repairing athymic mice skin defects than common tissue-engineered skin without KGF nanocapsules or skin auto-graft.

Animals ; Cell Culture Techniques ; Cells, Cultured ; Dermatologic Surgical Procedures ; Dermis ; cytology ; Epidermis ; cytology ; Fibroblast Growth Factor 7 ; Fibroblasts ; cytology ; Humans ; Mice ; Mice, Nude ; Nanocapsules ; Skin ; cytology ; injuries ; Skin Transplantation ; Tissue Engineering ; methods ; Tissue Scaffolds