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

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 explore the role of cytokines and keratinocytes in the survival mechanism of mixed auto and allogeneic skin grafting.

METHODSThirty-six SD rats were employed in the study. The rat model with mixed auto and allogeneic skin grafting and mixed human epithelial and lymphocytic culture (MELC) model were established. The change of IL-10 in the serum and the supernatant of the cultured tissue sample from the local wound was observed after the mixed skin grafting in scalded rats. And the role of epithelium in the induction of immunosuppression in vitro was monitored.

RESULTSThe serum IL-10 content in the rats with mixed skin grafting (25.89 +/- 2.82 ng/L) at 7 postoperative day (POD) was evidently higher than that in normal rats (14.20 +/- 2.43 ng/L) (P < 0.05). The IL-10 content in the culture supernatant of rat tissue samples exhibited evident different during 4-14 PODs (P < 0.05-0.01), while which was no difference to that in normal rat on 21st and 28th POD. The inhibiting effects of autologous epithelia and keratinocytes in MELC system were correlated with their dosage. After the adding of autologous keratinocytes to MELC system the cytokines secreted from Th1 could induce the secretion of cytokines from Th2 by IL-10 mediation. This effect could be corrected by the addition of monoclonal antibody of IL-10.

CONCLUSIONThe keratinocytes inlayed in the autoskin during mixed grafting could increase the local IL-10 level by activating Th2 cells, which might be one of the important reasons of the survival of mixed skin grafting.

Animals ; Burns ; immunology ; surgery ; Cytokines ; metabolism ; Giant Cells, Langhans ; cytology ; Graft Survival ; immunology ; Humans ; Interleukin-10 ; metabolism ; Keratinocytes ; cytology ; Lymphocyte Culture Test, Mixed ; Male ; Rats ; Rats, Sprague-Dawley ; Skin Transplantation ; immunology ; Th2 Cells ; metabolism ; Transplantation, Autologous ; Transplantation, Homologous

OBJECTIVETo investigate the epithelial growth factor (EGF) expression of EGF gene-transfected keratinocytes and its effect on cell proliferation after grafting.

METHODSNewborn Balb/c mouse keratinocytes and gene transfected keratinocytes were seeded on the surface of acellular dermal matrix and cocultured in different ratios as follows: 1:1, 1:3, or 1:5 1 week after culture. The composite skin was grafted onto the full-thickness wound in Balb/c mouse. Specimen was harvested at interval after grafting and underwent the immunohistochemistry staining for EGF and proliferating cell nuclear antigen (PCNA).

RESULTSImmunohistochemical staining showed EGF was expressed in the newly generated epidermis 1-2 week after grafting of the composite skin comprising Balb/c mouse keratinocytes and gene-transfected keratinocytes (at the ratio of 1:5). One week after surgery, Anti-PCNA positive basal cells were more than that in composite skin containing Balb/c mouse keratinocytes alone (P<0.01).

CONCLUSIONThe gene-transfected keratinocytes expresses EGF and promotes the proliferation of keratinocytes in the early stage after transplantation.

Animals ; Animals, Newborn ; Cell Proliferation ; Cells, Cultured ; Coculture Techniques ; Epidermal Growth Factor ; biosynthesis ; genetics ; Keratinocytes ; cytology ; metabolism ; transplantation ; Mice ; Mice, Inbred BALB C ; Skin ; injuries ; Skin Transplantation ; Tissue Engineering ; Transfection

OBJECTIVETo investigate the practicability of repair of full skin loss of rabbits with composite chitosan artificial skin.

METHODSDermal substitute was prepared aseptically by mixing fibroblasts with composite dermal matrix gel. Keratinocytes were then seeded on the substitute and submersion - cultured thereafter for 1 week in keratinocyte culture medium. The composite was further cultured for 1 approximately 2 weeks on the surface of the culture liquid to form artificial skin. The composite chitosan artificial skin was then grafted onto the full skin loss wound of rabbits. Histological changes were undertaken periodically by tissue sampling from the grafted wound. The systemic reaction of rabbits to the artificial skin was observed.

RESULTSAll the grafted wounds healed very well without any suppuration, bleeding or infection under the grafted skin. No obvious immune rejection was seen. The artificial skin could cover the wounds for a long time with good elasticity and easy to be manipulation.

CONCLUSIONThe composite chitosan artificial skin could be an optimal biological dressing with good histocompatibility and easy to be manipulation.

Animals ; Burns ; surgery ; Cells, Cultured ; Chitin ; analogs & derivatives ; metabolism ; Chitosan ; Dermatologic Surgical Procedures ; Humans ; Keratinocytes ; cytology ; metabolism ; Male ; Rabbits ; Skin ; injuries ; Skin Transplantation ; methods ; Skin, Artificial ; Transplantation, Heterologous ; Wound Healing

OBJECTIVETo observe the effectiveness of prevention and cure for maxillary growth deformity following tissue engineered oral mucosa implantation on mucoperiosteal denuded palate process in young rat.

METHODSHard palate mucoperiosteum of a SD baby rat were excised and oral keratinocytes were isolated and cultured. Tissue engineered oral mucosa was fabricated with the cultured oral keratinocytes and the membrane made of sodium alginate (SA). 80 female three-week-old SD rats were used as subjects in this study. The animals were divided randomly into a normal control group and 3 experimental groups, each group included 20 rats. Normal control group (NG) were not operated. Hard palate mucoperiosteum on left side in all experimental groups were excised, exposed bone were not treated in denuded group (DG), but repaired with membrane in material group (MG) and repaired with the tissue engineered oral mucosa in mucosal group (MUG). All the animals were sacrificed at 9th week postoperatively (12 weeks old), and the clean widths of right and left hard palatal were measured under a dissection microscope. The difference between palatal widths of two sides and the asymmetry ratio between the different groups were compared and analyzed.

RESULTSNo significant difference in asymmetry was discovered between the DG and the MG, but the asymmetry in MUG was less than DG or MG.

CONCLUSIONTissue engineered oral mucosal implantation in palatoplasty is an effective method in preventing and curing secondary maxilla deformity by repairing denuded bone wound.

Animals ; Animals, Newborn ; Cleft Palate ; physiopathology ; surgery ; Keratinocytes ; cytology ; Maxilla ; growth & development ; Mouth Mucosa ; transplantation ; Oral Surgical Procedures ; methods ; Palate, Hard ; surgery ; Periosteum ; surgery ; Rats ; Rats, Sprague-Dawley ; Tissue Engineering

Skin is the largest organ in human system and plays a vital role as a barrier against environment and pathogens. Skin regeneration is important in tissue engineering especially in cases of chronic wounds. With the tissue engineering technology, these skins equivalent have been use clinically to repair burns and wounds. Consented redundant skin samples were obtained from patients aged 9 to 65 years old. Skin samples were digested with dispase, thus separating the epidermis and the dermis layer. The epidermis layer was trypsinized and cultured in DKSFM in 6-well plate at 37 degrees C and 5% CO2. Once confluent, the culture were trypsinized and the cells were pooled. Cells were counted using haemacytometer. Doubling time and viability were calculated and analysed. From the result, we conclude that doubling time and viability of in vitro keratinocytes cultured in DKSFM media is not age dependant.
Age Factors ; Burns/physiopathology ; Burns/*therapy ; Cell Aging/*physiology ; Cell Division/physiology ; Cell Survival/physiology ; Chronic Disease ; Keratinocytes/*cytology ; *Skin Transplantation ; Statistics ; Tissue Engineering/*methods ; Wound Healing/physiology ; Wounds and Injuries/physiopathology ; Wounds and Injuries/*therapy